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1.
Infect Disord Drug Targets ; 20(4): 423-432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30950360

RESUMO

Viral interference, originally, referred to a state of temporary immunity, is a state whereby infection with a virus limits replication or production of a second infecting virus. However, replication of a second virus could also be dominant over the first virus. In fact, dominance can alternate between the two viruses. Expression of type I interferon genes is many times upregulated in infected epithelial cells. Since the interferon system can control most, if not all, virus infections in the absence of adaptive immunity, it was proposed that viral induction of a nonspecific localized temporary state of immunity may provide a strategy to control viral infections. Clinical observations also support such a theory, which gave credence to the development of superinfection therapy (SIT). SIT is an innovative therapeutic approach where a non-pathogenic virus is used to infect patients harboring a pathogenic virus. For the functional cure of persistent viral infections and for the development of broad- spectrum antivirals against emerging viruses a paradigm shift was recently proposed. Instead of the virus, the therapy should be directed at the host. Such a host-directed-therapy (HDT) strategy could be the activation of endogenous innate immune response via toll-like receptors (TLRs). Superinfection therapy is such a host-directed-therapy, which has been validated in patients infected with two completely different viruses, the hepatitis B (DNA), and hepatitis C (RNA) viruses. SIT exerts post-infection interference via the constant presence of an attenuated non-pathogenic avian double- stranded (ds) RNA viral vector which boosts the endogenous innate (IFN) response. SIT could, therefore, be developed into a biological platform for a new "one drug, multiple bugs" broad-spectrum antiviral treatment approach.


Assuntos
Antivirais/farmacologia , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/farmacologia , Vírus de RNA/fisiologia , Interferência Viral/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Expressão Gênica , Humanos , Interferon Tipo I/isolamento & purificação , Vírus de RNA/imunologia , Carga Viral , Replicação Viral/efeitos dos fármacos
2.
Hepatol Med Policy ; 3: 2, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30288325

RESUMO

Background: Viral hepatitis deaths from acute infection, cirrhosis, and liver cancer have risen from the tenth to the seventh leading cause of death worldwide between 1990 and 2013. Even in the oral direct acting antiviral (DAA) agent era there are still large numbers of patients with unmet needs. Medications approved for treatment of chronic hepatitis B virus (HBV) infection do not eradicate HBV often requiring treatment for life associated with risks of adverse reactions, drug resistance, nonadherence, and increased cost. Although DAAs increased virologic cure rates well over 90% in all hepatitis C virus (HCV) genotypes, HCV infection still cannot be cured in a small but significant minority of patients. While most of the medical issues of HCV treatment have been solved, the current costs of DAAs are prohibitive. Results: The post-infection viral superinfection treatment (SIT) platform technology has been clinically proven to be safe and effective to resolve acute and persistent viral infections in 42 HBV and HCV patients (20 HBV, 22 HCV), and in 4 decompensated patients (2 HBV, 2 HCV). SIT employs a non-pathogenic avian double stranded RNA (dsRNA) virus, a potent activator of antiviral gene responses. Unexpectedly, SIT is active against unrelated DNA (HBV) and RNA (HCV) viruses. SIT does not require lifelong therapy, which is a major advantage considering present HBV treatments. The new viral drug candidate (R903/78) is homogeneously produced by reverse genetics in Vero cells. R903/78 has exceptional pH and temperature stability and also excellent long-term stability; therefore, it can be orally administered, stored and shipped without freezing. Since R903/78 is easy to stockpile, the post-infection SIT could also alleviate the logistic hurdles of surge capacity in vaccine production during viral pandemics. Conclusion: To help large number of HBV and HCV patients with unmet needs, broad-spectrum antiviral drugs effective against whole classes of viruses are urgently needed. The innovative SIT technological platform will be a great additional armament to conquer viral hepatitis, which is still a major cause of death and disability worldwide.

3.
J Gene Med ; 17(6-7): 116-31, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25929556

RESUMO

BACKGROUND: Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained. METHODS: To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively. RESULTS: Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro. CONCLUSIONS: Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use.


Assuntos
Vírus da Doença Infecciosa da Bursa , Superinfecção/terapia , Administração Oral , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Linhagem Celular , Hepatite B/imunologia , Hepatite B/terapia , Hepatite C/imunologia , Hepatite C/terapia , Humanos , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/imunologia , Camundongos , Genética Reversa , Superinfecção/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
4.
PLoS One ; 7(10): e46981, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056548

RESUMO

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.


Assuntos
Adenoviridae/genética , Antígenos de Superfície/genética , Antígenos CD40/metabolismo , Células Dendríticas/imunologia , Vetores Genéticos/genética , Glutamato Carboxipeptidase II/genética , Neoplasias da Próstata/prevenção & controle , Vacinação/métodos , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adjuvantes Imunológicos/metabolismo , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Antígenos de Superfície/metabolismo , Antígenos CD40/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Glutamato Carboxipeptidase II/metabolismo , Antígenos HLA-A/genética , Humanos , Interferon gama/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia
5.
PLoS One ; 6(2): e16792, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21347423

RESUMO

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of (99m)Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a (99m)Tc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.


Assuntos
Adenoviridae/genética , Fusão Gênica Artificial/métodos , Proteínas do Capsídeo/genética , Metalotioneína/genética , Tomografia Computadorizada de Emissão de Fóton Único , Adenoviridae/metabolismo , Adenoviridae/fisiologia , Animais , Ligação Competitiva , Replicação do DNA , DNA Viral/biossíntese , Feminino , Vetores Genéticos/genética , Células HEK293 , Humanos , Metais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Compostos de Organotecnécio/metabolismo , Estabilidade Proteica , Vírion/genética , Vírion/metabolismo , Vírion/fisiologia
6.
J Virol ; 85(3): 1408-14, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21106739

RESUMO

The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector.


Assuntos
Antígenos Virais/genética , Portadores de Fármacos , Epitopos/genética , Vetores Genéticos , Hepacivirus/genética , Vírus da Doença Infecciosa da Bursa/genética , Vacinas contra Hepatite Viral/genética , Animais , Antígenos Virais/imunologia , Linhagem Celular , Chlorocebus aethiops , Epitopos/imunologia , Hepacivirus/imunologia , Humanos , Doenças da Íris , Mutagênese Insercional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Deleção de Sequência , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas contra Hepatite Viral/imunologia
7.
Viruses ; 2(8): 1681-703, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21994701

RESUMO

Adenovirus (Ad) vectors, in particular those of the serotype 5, are highly attractive for a wide range of gene therapy, vaccine and virotherapy applications (as discussed in further detail in this issue). Wild type Ad5 virus can replicate in numerous tissue types but to use Ad vectors for therapeutic purposes the viral genome requires modification. In particular, if the viral genome is modified in such a way that the viral life cycle is interfered with, a specific producer cell line is required to provide trans-complementation to overcome the modification and allow viral production. This can occur in two ways; use of a producer cell line that contains specific adenoviral sequences incorporated into the cell genome to trans-complement, or use of a producer cell line that naturally complements for the modified Ad vector genome. This review concentrates on producer cell lines that complement non-replicating adenoviral vectors, starting with the historical HEK293 cell line developed in 1977 for first generation Ad vectors. In addition the problem of replication-competent adenovirus (RCA) contamination in viral preparations from HEK293 cells is addressed leading to the development of alternate cell lines. Furthermore novel cell lines for more complex Ad vectors and alternate serotype Ad vectors are discussed.

8.
J Gene Med ; 10(2): 123-31, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18064718

RESUMO

BACKGROUND: The kinetics of gene expression from adenovirus-based delivery vectors will be an important variable influencing the efficacy and toxicity of these vectors. As different promoters have variable strengths and kinetic profiles, the optimal dose of a therapeutic transgene product over time may be achieved by varying the promoter. METHODS: We analyzed several viral and cellular promoters in the context of adenovector gene delivery in the mouse. The kinetics of transgene expression was evaluated following intramuscular and intravenous delivery. RESULTS: Transgene expression from the cytomegalovirus (CMV) promoter was rapidly down-regulated in the tissues following intravenous administration of adenovectors. In contrast, transgene expression from the Rous sarcoma virus (RSV) promoter increased over time such that, at 3 weeks, expression was 10-fold higher than that from the CMV promoter-containing vector in all tissues. The kinetics of transgene expression from these vectors was similar when they were delivered via the intramuscular route in BALB/c, C57BL/6 and immunodeficient mice. Efficient repeat administration of an adenovirus vector, in the presence of neutralizing antibodies, was achieved in the skeletal muscle and transgene expression persisted with the same kinetics as in naïve animals. CONCLUSIONS: These results demonstrate that the in vivo kinetics of transgene expression by adenovectors is greatly influenced by the promoter. Adenovectors can be designed to deliver a transient bolus or a sustained level of protein expression in the target tissue depending on the requirements for particular indications. These results have implications for both therapeutic and vaccine indications.


Assuntos
Adenoviridae/genética , Regulação da Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Regiões Promotoras Genéticas/genética , Transgenes/genética , Animais , Linhagem Celular , Citomegalovirus/genética , Feminino , Humanos , Imunidade Celular , Imunocompetência , Injeções Intravenosas , Cinética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Esquelético/metabolismo , Vírus do Sarcoma de Rous/genética , Fatores de Tempo , Transdução Genética
9.
Mol Biotechnol ; 35(3): 263-73, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17652790

RESUMO

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.


Assuntos
Adenoviridae/genética , Vetores Genéticos , Transgenes , Vacinas Virais/genética , Adenoviridae/fisiologia , Sequência de Bases , Linhagem Celular , Primers do DNA , DNA Viral/genética , DNA Viral/isolamento & purificação , Humanos , Replicação Viral
10.
J Virol ; 80(11): 5523-30, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16699033

RESUMO

On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo.


Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/química , Vetores Genéticos/química , Adenoviridae/genética , Animais , Proteínas do Capsídeo/genética , Epitopos/genética , Epitopos/imunologia , Vetores Genéticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Virais
11.
Cancer Immunol Immunother ; 55(11): 1412-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16612598

RESUMO

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdDelta24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdDelta24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.


Assuntos
Adenoviridae/genética , Vetores Genéticos , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Capsídeo/química , Humanos , Modelos Biológicos , Neoplasias/genética
12.
J Clin Invest ; 115(5): 1281-9, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15841217

RESUMO

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.


Assuntos
Adenoviridae , Epitopos/imunologia , Vetores Genéticos , Porinas/imunologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Haplótipos , Antígenos de Histocompatibilidade/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Dados de Sequência Molecular , Porinas/genética , Estrutura Secundária de Proteína , Infecções por Pseudomonas/imunologia
13.
J Gene Med ; 6(9): 992-1002, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15352072

RESUMO

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.


Assuntos
Terapia Genética/métodos , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade/terapia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Vetores Genéticos/uso terapêutico , Humanos , Recém-Nascido , Ratos , Retina/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Corpo Vítreo/metabolismo
14.
J Gene Med ; 6(3): 309-16, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15026992

RESUMO

BACKGROUND: Modification of the fiber proteins in replication-deficient adenoviral (Ad) vectors through incorporation of specific receptor-binding motifs may represent a strategy to enhance their tissue targeting capabilities. METHODS: In this study, we compared an unmodified Ad (GV10) with two mutated vectors obtained by insertion of specific target sequences that redirect binding, either toward alpha(V) integrin (RGD) or heparan sulfate (UTV) cellular receptors, for reporter gene expression spatial distribution in the rabbit skeletal muscle. In a first series of experiments, injection volume was kept constant and activity of a lacZ transgene was evaluated 48 h after injection of the Ad vectors at different doses. In separate experiments, the effects of different volumes of injection at a constant dose of Ad vector were monitored. RESULTS: All vectors evaluated showed a significant increase in the number of lacZ-positive muscle segments, with increasing vector dose. However, in muscles treated with the UTV vector, fewer muscle fibers were beta-gal-positive than in GV10 or RGD vector treated animals. In fact, total beta-gal activity increased in a dose-dependent fashion in the GV10- and RGD-treated muscles, but not in the UTV-treated ones. Remarkably, in samples from UTV-treated animals, a volume-dependent enhancement of transgene expression was observed during experiments performed at the same dose and different injection volumes. CONCLUSIONS: The results of the present study demonstrate that altering Ad affinity for cellular receptors modulates the level and distribution of transgene activity, conferring characteristics that may allow for treatment customization.


Assuntos
Adenoviridae/genética , Vetores Genéticos , Heparitina Sulfato/metabolismo , Integrina alfaV/metabolismo , Transgenes/genética , Animais , Feminino , Genes Reporter , Vetores Genéticos/administração & dosagem , Proteoglicanas de Heparan Sulfato/metabolismo , Imunoquímica , Injeções Intramusculares , Músculo Esquelético/metabolismo , Músculo Esquelético/virologia , Coelhos , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Vírion/metabolismo , beta-Galactosidase/análise , beta-Galactosidase/genética
15.
Int J Mol Med ; 13(4): 581-7, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15010860

RESUMO

We have studied an age-related impairment in angiogenesis and evaluated the effect of overexpressing VEGF in this situation. Polyvinyl alcohol sponges were implanted subcutaneously into aged (24-month), adult (12-month), and young (2-month) rats. Blood vessel ingrowth and proliferative activity in the sponges were assessed by histology with immunostaining for von Willebrand's factor and proliferating cell nuclear antigen (PCNA), respectively. The percentage of total sponge area filled with ingrowing fibrovascular tissue was minimal in aged rats, intermediate in adult rats and highest in young rats. A similar pattern was observed for the total blood vessel numbers in the sponges from old to young animals. The percentage of total sponge endothelial cells (ECs) showing proliferative activity (PCNA positive) was lowest in the aged animals, intermediate in the adult rats and highest in the young rats. To further explore the mechanism of impaired angiogenesis in aged animals, we investigated and found a reduced level of endogenous VEGF protein expression in 12-month-old rats compared to that in 2-month-old rats. VEGF121 gene transfer significantly enhanced blood vessel and fibrovascular tissue ingrowth in adult/aged rats. Adenoviral-VEGF gene transfer also significantly stimulated EC proliferation in aged and adult rats. However, identical treatment failed to further stimulate the already more robust angiogenesis in young animals. The different angiogenic response in adult vs. young rats was not due to differences in gene transfer efficiency, since similar levels of human VEGF121 protein was detected in adult and young rats. Our results indicate that the decreased angiogenic response with aging is associated with reduced EC proliferation and reduced endogenous VEGF production. Adenoviral-VEGF121 gene transfer is effective in augmenting angiogenesis, particularly in older animals.


Assuntos
Adenoviridae/genética , Envelhecimento , Técnicas de Transferência de Genes , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Northern Blotting , Divisão Celular , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imuno-Histoquímica , Neovascularização Patológica , Ratos , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
16.
Cancer Res ; 64(4): 1386-95, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14973054

RESUMO

Nitric oxide is a potent radiosensitizer of tumors, but its use clinically is limited by serious side effects when administered systemically. We have demonstrated previously that gene transfer of the inducible nitric oxide synthase gene (iNOS) into colorectal cancer cells enhances radiation-induced apoptosis in vitro. The objectives of this study were to further characterize the effects of iNOS gene transfer on the radiosensitivity of human colorectal cancer cells in vitro and tumors grown in athymic nude mice. Adenoviral gene transfer of iNOS (AdiNOS) into human colorectal cancer cell lines (HCT-116 and SNU-1040 cells) significantly enhanced the effects of radiation with sensitizing enhancement ratios (0.1) of 1.65 and 1.6, respectively. The radiation enhancement induced by iNOS was associated with increased iNOS expression and nitric oxide production and prevented by L-NIO, an enzymatic inhibitor of iNOS. AdiNOS treatment of HCT-116 tumors combined with radiation (2 Gy x three fractions) led to a 3.4-fold greater (P < 0.005) tumor growth delay compared with radiation (RT) alone. AdiNOS plus RT also caused significant (P < 0.01) tumor regression with 63% of tumors regressing compared with only 6% of tumors treated with RT. AdiNOS plus RT significantly (P < or = 0.001) increased the percentage of apoptotic cells (22 +/- 4%) compared with either tumors treated with control vector plus RT (9 +/- 1%), AdiNOS alone (9 +/- 3%), or no treatment (2 +/- 1%). These radiosensitizing effects of AdiNOS occurred at low infection efficiency (4% of tumor infected), indicating a significant bystander effect.


Assuntos
Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/radioterapia , Terapia Genética , Óxido Nítrico Sintase/genética , Tolerância a Radiação , Adenoviridae/genética , Animais , Apoptose , Ciclo Celular , Neoplasias Colorretais/patologia , Transferência Genética Horizontal , Humanos , Camundongos , Camundongos Nus , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo II
17.
Am J Physiol Heart Circ Physiol ; 286(2): H570-4, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14551046

RESUMO

Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 10(9) plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or beta-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10(-5) to 10(-9) M), endothelium-dependent relaxation to acetylcholine (10(-5) to 10(-9) M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.


Assuntos
Biopterina/análogos & derivados , Artérias Carótidas/fisiologia , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Acetilcolina/farmacologia , Adenoviridae , Animais , Disponibilidade Biológica , Biopterina/farmacocinética , Artérias Carótidas/efeitos dos fármacos , Endotélio Vascular/fisiologia , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos , Humanos , Indometacina/farmacologia , Masculino , Neopterina/metabolismo , Papaverina/farmacologia , Fenilefrina/farmacologia , Coelhos , Proteínas Recombinantes/metabolismo
18.
J Gen Virol ; 84(Pt 12): 3417-3422, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14645922

RESUMO

A method was developed to generate a complex cDNA expression library within an adenovirus type 5 (Ad5)-based vector backbone, termed AdLibrary. Construction of the AdLibrary entailed the conversion of an Ad5 genome-containing cosmid to infectious virus particles. The Ad5 genome was modified by replacing the E1A and E1B genes with a Rous sarcoma virus-driven expression cassette. Conversion was accomplished by liberating the viral genome by restriction enzyme digestion and transfection in HEK 293 cells, which support the growth of E1A/E1B-deficient virus. A test AdLibrary demonstrated the possibility of converting and identifying a marker gene present at a frequency of 1/10(5) in the cosmid library. To demonstrate the utility of this technology, an AdLibrary was used to isolate a viral gene by its biological function. Virus growth was selected for with an AdLibrary on A549 cells, which do not complement for E1A/E1B function. The AdLibrary was generated with cDNAs derived from HeLa cells productively infected with Ad5. A cDNA corresponding to Ad5 E1A 13S was selected and isolated from the AdLibrary using this strategy. Since multiple genes are assayed simultaneously, this technology should expedite the discovery of genes affecting defined biological activities. This AdLibrary approach provides an opportunity to exploit the efficient gene delivery capabilities of adenovirus vectors for the rapid discovery of gene and protein function.


Assuntos
Adenoviridae/genética , Biblioteca Gênica , Genoma Viral , Adenoviridae/química , Proteínas E1 de Adenovirus/fisiologia , Linhagem Celular , DNA Complementar , DNA Viral , Teste de Complementação Genética , Vetores Genéticos , Humanos
19.
Circulation ; 108(10): 1238-45, 2003 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-12925450

RESUMO

BACKGROUND: We recently reported that arterial superoxide (O2-) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2--induced vascular dysfunction. However, the interaction between O2- and BH4 on endothelial function and the underlying mechanisms are unknown. METHODS AND RESULTS: The present study tested the hypothesis that BH4 deficiency due to ET-1-induced O2- leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5'-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2- levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2- and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2- and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. CONCLUSIONS: These results indicate that a BH4 deficiency resulting from ET-1-induced O2- via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2- in low renin mineralocorticoid hypertension.


Assuntos
Biopterina/análogos & derivados , Biopterina/metabolismo , Endotélio Vascular/fisiopatologia , GTP Cicloidrolase/genética , Terapia Genética/métodos , Hipertensão/terapia , Acetofenonas/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Atrasentana , Biopterina/deficiência , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiopatologia , Óxidos N-Cíclicos/uso terapêutico , Desoxicorticosterona , Modelos Animais de Doenças , Antagonistas dos Receptores de Endotelina , Endotelina-1/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , GTP Cicloidrolase/metabolismo , GTP Cicloidrolase/farmacologia , Técnicas de Transferência de Genes , Humanos , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Óxido Nítrico/metabolismo , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A , Cloreto de Sódio , Marcadores de Spin , Superóxidos/metabolismo , Vasodilatação/efeitos dos fármacos
20.
Mol Ther ; 7(5 Pt 1): 597-603, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12718902

RESUMO

In this study the effect of local adenoviral-mediated delivery of inducible nitric oxide synthase on restenosis was evaluated in a porcine coronary stented model. Local gene transfer of recombinant adenoviral vectors that encode human inducible nitric oxide synthase (AdiNOS) was tested. Control vector (AdNull) lacked a recombinant transgene. Endoluminal delivery of 1.0 x 10(11) adenoviral particles was accomplished in 45 s using the Infiltrator catheter (Interventional Technologies, San Diego, CA). Coronary stents were deployed, oversized by a ratio of 1.2:1, in the treated segments immediately after gene transfer. Fourteen animals were sacrificed at day 28 to evaluate the effects of iNOS gene transfer on morphometric indices, and 4 animals were sacrificed at day 4 for detection of human iNOS expression by RT-PCR. iNOS mRNA was detected in six of eight iNOS-transferred arteries, whereas no expression of human iNOS was detected in the nontarget arteries. Morphometric analysis showed that iNOS transfer significantly reduced neointimal formation (3.41 +/- 1.12 mm(2) vs 2.14 +/- 0.68 mm(2), P < 0.05). We concluded that efficient intramural adenovirus-mediated iNOS transfer can be achieved by using Infiltrator catheters. iNOS gene transfer significantly reduces neointimal hyperplasia following stent injury.


Assuntos
Adenoviridae/genética , Arteriopatias Oclusivas/terapia , Terapia Genética , Óxido Nítrico Sintase/genética , Túnica Íntima/patologia , Animais , Arteriopatias Oclusivas/enzimologia , Arteriopatias Oclusivas/patologia , Estenose Coronária/enzimologia , Estenose Coronária/patologia , Estenose Coronária/terapia , Vasos Coronários/patologia , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Técnicas Imunoenzimáticas , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Stents , Suínos , Porco Miniatura , Transfecção
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