RESUMO
We have previously reported that recombinant adeno-associated virus serotype 3 (AAV3) vectors transduce human liver tumors more efficiently in a mouse xenograft model following systemic administration. Others have utilized AAV8 vectors expressing miR-26a and miR-122 to achieve near total inhibition of growth of mouse liver tumors. Since AAV3 vectors transduce human hepatic cells more efficiently than AAV8 vectors, in the present studies, we wished to evaluate the efficacy of AAV3-miR-26a/122 vectors in suppressing the growth of human hepatocellular carcinoma (HCC) cells in vitro, and human liver tumors in a mouse model in vivo. To this end, a human HCC cell line, Huh7, was transduced with various multiplicities of infection (MOIs) of AAV3-miR-26a or scAAV3-miR-122 vectors, or both, which also co-expressed a Gaussia luciferase (GLuc) reporter gene. Only a modest level of dose-dependent growth inhibition of Huh7 cells (~12-13%) was observed at the highest MOI (1 × 105 vgs/cell) with each vector. When Huh7 cells were co-transduced with both vectors, the extent of growth inhibition was additive (~26%). However, AAV3-miR-26a and scAAV3-miR-122 vectors led to ~70% inhibition of growth of Huh-derived human liver tumors in a mouse xenograft model in vivo. Thus, the combined use of miR-26a and scAAV3-miR-122 delivered by AAV3 vectors offers a potentially useful approach to target human liver tumors.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Proliferação de Células , Dependovirus/genética , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Camundongos , MicroRNAs/genética , SorogrupoRESUMO
We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to 1/2 binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (1) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (2) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (3) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (4) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo; and (5) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE 1/2 binding site, and insertion of a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y AAV vectors, which are serotype, transgene, or promoter agnostic, should be useful in human gene therapy.
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The first generation of adeno-associated virus (AAV) vectors composed of the naturally occurring capsids and genomes, although effective in some instances, are unlikely to be optimal for gene therapy in humans. The use of the first generation of two different AAV serotype vectors (AAV9 and AAVrh74) in four separate clinical trials failed to be effective in patients with Duchenne muscular dystrophy, although some efficacy was observed in a subset of patients with AAVrh74 vectors leading to US Food and Drug Administration approval (Elevidys). In two trials with the first generation of AAV9 vectors, several serious adverse events were observed, including the death of a patient in one trial, and more recently, in the death of a second patient in an N-of-1 clinical trial. In a fourth trial with the first generation of AAVrh74 vectors, myositis and myocarditis were also observed. Here, we report that capsid- and genome-modified optimized AAVrh74 vectors are significantly more efficient in transducing primary human skeletal muscle cells in vitro and in all major muscle tissues in vivo following systemic administration in a murine model. The availability of optimized AAVrh74 vectors promises to be safe and effective in the potential gene therapy of muscle diseases in humans.
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Recombinant AAV serotype vectors and their variants have been or are currently being used for gene therapy for hemophilia in several phase I/II/III clinical trials in humans. However, none of these trials have included children with hemophilia since the traditional liver-directed AAV gene therapy will not work in these patients because of the following reasons: (i) Up until age 10-12, the liver is still growing and dividing, and with every cell division, the AAV vector genomes will be diluted out due to their episomal nature; and (ii) Repeated gene delivery will be needed, but repeat dosing, even with an ideal AAV vector is not an option because of pre-existing antibodies to AAV vectors following the first administration. Here we describe the development of an optimized human Factor IX (hF.IX) gene expression cassette under the control of a human liver-specific transthyretin promoter covalently flanked by AAV inverted terminal repeats (ITRs) with no open ends (optNE-TTR-hF.IX), which mediated ~sixfold higher hF.IX levels than that from a linear TTR-hF.IX DNA construct in human hepatoma cells up to four-weeks post-transfection. In future studies, encapsidation of the optNE-TTR-hF.IX DNA in liver-targeted synthetic liposomes, may provide a viable approach for the potential gene therapy for hemophilia in children.
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Metal elements are essential components of approximately half of all cellular proteins, and approximately one-third of all known enzymes thus far are metalloenzymes. Several cellular proteins and enzymes undoubtedly impact the transduction efficiency of recombinant adeno-associated virus (AAV) vectors, but the precise role of metal ions in this process has not been studied in detail. In the present studies, we systematically evaluated the effects of all 10 essential metal ions (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium, and zinc) on the transduction efficiency of AAV vectors. We report herein that five essential metal ions (iron, magnesium, manganese, molybdenum, and sodium) had little to no effect, and calcium strongly inhibited the transduction efficiency of AAV2 vectors. Whereas copper and potassium increased the transduction efficiency by â¼5-fold and â¼2-fold, respectively, at low concentrations, both essential metals were strongly inhibitory at higher concentrations. Calcium also inhibited the transduction efficiency by â¼3-fold. Two metal ions (cobalt and zinc) increased the transduction efficiency up to â¼10-fold in a dose-dependent manner. The combined use of cobalt and zinc resulted in more than an additive effect on AAV2 vector transduction efficiency (â¼30-fold). The transduction efficiency of AAV serotypes 1 through 6 (AAV1-AAV6) vectors was also augmented by zinc. Similarly, the transduction of both single-stranded (ss) and self-complementary (sc) AAV3 vectors was enhanced by zinc. Zinc treatment also led to a dose-dependent increase in expression of a therapeutic protein, the human clotting factor IX (hF.IX), mediated by scAAV3 vectors in a human hepatic cell line. This simple strategy of essential metal ion-mediated enhancement may be useful to lower the dose of AAV vectors for their optimal use in human gene therapy.
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A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by â¼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.
Assuntos
Dependovirus/genética , Antígenos de Histocompatibilidade Classe II/genética , Imunidade Humoral , Sequências Repetidas Terminais , Animais , DNA Viral , Regulação para Baixo , Regulação da Expressão Gênica , Genes MHC da Classe II , Terapia Genética , Vetores Genéticos , Células HEK293 , Antígenos HLA/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Fator Regulador X1/genética , Homologia de SequênciaRESUMO
We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro, as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as ß-thalassemia and sickle cell disease.
RESUMO
Recombinant adeno-associated virus 2 (AAV) vectors encapsidate single-stranded genomes of either polarity equally frequently in separate mature virions. Because viral genomes of either polarity are transcriptionally inactive, both the failure to undergo viral second-strand DNA synthesis and the failure to undergo DNA strand annealing have been proposed as possible reasons to account for the observed low efficiency of transgene expression. We compared the transduction efficiencies of conventional AAV vectors containing both [-] and [+] polarity genomes with those containing either the [-] or the [+] polarity genomes, in vitro as well as in vivo. We document that the transduction efficiency of single-polarity AAV vectors is significantly enhanced by (i) co-infection with adenovirus; (ii) small interfering RNA (siRNA)-mediated down-modulation of a cellular protein, FKBP52, tyrosine-phosphorylated forms of which inhibit AAV second-strand DNA synthesis; (iii) over-expression of a cellular protein tyrosine phosphatase, T cell protein tyrosine phosphatase (TC-PTP), which catalyzes tyrosine-dephosphorylation of FKBP52; and (iv) deliberate over-expression of TC-PTP, or the absence of FKBP52, respectively, in TC-PTP-transgenic mice and in FKBP52-knockout mice. These data confirm that viral second-strand DNA synthesis, rather than DNA strand annealing, is the rate-limiting step in efficient transduction by AAV vectors. This finding has implications in the use of these vectors in human gene therapy.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Transgenes/genética , Animais , Northern Blotting , Southern Blotting , Linhagem Celular Tumoral , Células HeLa , Humanos , Camundongos , Modelos Teóricos , RNA Interferente Pequeno/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/fisiologia , Transdução GenéticaRESUMO
Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT's short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.
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Cocaine- and amphetamine-regulated transcript (CART) peptide is a neuro-peptide implicated in the regulation of energy homeostasis. CART mRNA and its encoded peptide have been found in many brain regions that regulate energy balance. To investigate the effects of chronic central expression of CART in the diet-induced-obese (DIO) rats, we constructed and packaged recombinant adeno-associated virus (AAV) 2 vector containing rat CART cDNA and a reporter gene encoding green fluorescence protein (AAV-rCART-hrGFP) driven by the CMV promoter. Approximately 1x10(11) particles of AAV-rCART-hrGFP or control vector AAV-IRES-hrGFP (AAV-hrGFP in short) were injected through intracerebroventricular (ICV) cannulas into adult male DIO Long-Evans rats. Throughout the 7-month study period, AAV-rCART-hrGFP-injected rats had a significantly decreased food intake and body weight gain when compared with those of AAV-hrGFP-injected rats. AAV-rCART-hrGFP injection also modulated hyperphagia following a 24-hour fasting in these rats. Body composition analysis indicated that decreased body weight gain was due to reduction in lean body mass while fat mass was not affected. Expression of green fluorescence protein (GFP) suggested that recombinant AAV virions are located at cells surrounding the third ventricle and medial eminence. Our results demonstrate that chronic expression of CART in brain can modulate energy intake in diet-induced-obese rats. The CART pathway plays an important role in body mass regulation in DIO rats.
Assuntos
Vetores Genéticos/genética , Proteínas do Tecido Nervoso/fisiologia , Obesidade/fisiopatologia , Aumento de Peso/fisiologia , Animais , Composição Corporal/genética , Encéfalo/citologia , Encéfalo/metabolismo , Calorimetria , Dependovirus/genética , Dieta/efeitos adversos , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Injeções Intraventriculares , Masculino , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Obesidade/etiologia , Obesidade/genética , Ratos , Ratos Long-Evans , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Aumento de Peso/genéticaRESUMO
Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.
Assuntos
Dependovirus/genética , Vetores Genéticos/administração & dosagem , Células-Tronco Hematopoéticas/metabolismo , Proteínas Tirosina Fosfatases/genética , Células-Tronco/metabolismo , Transdução Genética , Animais , Ataxina-1 , Ataxinas , Células Cultivadas , DNA Recombinante/administração & dosagem , Dependovirus/classificação , Dependovirus/imunologia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/virologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Células-Tronco/virologia , Transgenes/fisiologiaRESUMO
Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.
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Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Células-Tronco Hematopoéticas/metabolismo , Transdução Genética/métodos , Animais , Linhagem Celular , Células Cultivadas , DNA Viral/análise , Feminino , Regulação da Expressão Gênica , Terapia Genética/métodos , Células-Tronco Hematopoéticas/virologia , Hidroxiureia/farmacologia , Óperon Lac/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Proteínas Recombinantes/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Vírion/fisiologiaRESUMO
Cathepsin S (Cat S) plays an important role in many pathological conditions, including abdominal aortic aneurysm (AAA). Inhibition of Cat S may provide a new treatment for AAA. To date, several classes of Cat S inhibitors have been reported, many of which form covalent interactions with the active site Cys25. Herein, we report the discovery of a novel series of noncovalent inhibitors of Cat S through a medium-throughput focused cassette screen and the optimization of the resulting hits. Structure-based optimization efforts led to Cat S inhibitors such as 5 and 9 with greatly improved potency and drug disposition properties. This series of compounds binds to the S2 and S3 subsites without interacting with the active site Cys25. On the basis of in vitro potency, selectivity, and efficacy in a CaCl2-induced AAA in vivo model, 5 (LY3000328) was selected for clinical development.
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We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.
Assuntos
Dependovirus/metabolismo , Vetores Genéticos/metabolismo , Chaperonas Moleculares/fisiologia , Animais , Transporte Biológico , Núcleo Celular/virologia , Células Cultivadas , Dependovirus/genética , Dineínas/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Heterozigoto , Camundongos , Camundongos Knockout , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Peso Molecular , Tacrolimo/metabolismo , Transdução GenéticaRESUMO
OBJECTIVE: To investigate stearoyl-coenzyme A desaturase (SCD) 1 expression in obesity-prone C57BL/6 mice and in obesity-resistant FVB mice to explore the relationship of SCD1 expression and susceptibility to diet-induced obesity. RESEARCH METHODS AND PROCEDURES: Nine-week-old C57BL/6 and FVB mice were fed either a high- or low-fat diet for 8 weeks. Body weight and body composition were measured before and at weeks 4 and 8 of the study. Energy expenditure was measured at weeks 1 and 5 of the study. Hepatic SCD1 mRNA was measured at 72 hours and at the end of study. Plasma leptin and insulin concentrations were measured at the end of study. RESULTS: When C57BL/6 mice were switched to a calorie-dense high-fat diet, animals gained significantly more body weight than those maintained on a low-calorie density diet primarily due to increased fat mass accretion. Fat mass continued to accrue throughout 8 weeks of study. Increased calorie intake did not account for all weight gain. On the high-fat diet, C57BL/6 mice decreased their energy expenditure when compared with mice fed a low-fat diet. In response to 8 weeks of a high-fat diet, SCD1 gene expression in liver increased >2-fold. In contrast, feeding a high-fat diet did not change body weight, energy expenditure, or SCD1 expression in FVB mice. DISCUSSION: Our study showed that a high-fat hypercaloric diet increased body adiposity first by producing hyperphagia and then by decreasing energy expenditure of mice susceptible to diet-induced obesity. Consumption of a high-fat diet in species predisposed to obesity selectively increased SCD1 gene expression in liver.
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Gorduras na Dieta/administração & dosagem , Expressão Gênica , Obesidade/genética , Proteínas/genética , Estearoil-CoA Dessaturase , Animais , Composição Corporal , Peso Corporal , Calorimetria Indireta , Ingestão de Alimentos , Ingestão de Energia , Metabolismo Energético , Predisposição Genética para Doença , Insulina/sangue , Leptina/sangue , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/enzimologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in approximately 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.
Assuntos
Dependovirus/genética , Genes Virais , Proteínas de Choque Térmico HSP90/metabolismo , Benzoquinonas , Western Blotting , Replicação do DNA , DNA Complementar/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Receptores ErbB/metabolismo , Terapia Genética , Células HeLa , Humanos , Lactamas Macrocíclicas , Modelos Biológicos , Oligonucleotídeos Antissenso/química , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Quinonas/farmacologia , Proteínas de Ligação a Tacrolimo/química , Transfecção , Transgenes , Tirosina/metabolismo , Tirfostinas/farmacologiaRESUMO
Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Vírus Auxiliares/genética , Proteínas Tirosina Fosfatases/genética , Transdução Genética/métodos , Animais , Linhagem Celular Tumoral , DNA Viral/análise , Terapia Genética/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Hepatócitos/química , Hepatócitos/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/metabolismoRESUMO
The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.