RESUMO
The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D-such as their cellular tropism, receptor usage, and in vivo biodistribution profile-remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)-a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.
Assuntos
Adenovírus Humanos/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Adenovírus Humanos/classificação , Adenovírus Humanos/metabolismo , Animais , Linhagem Celular , Genes Reporter , Terapia Genética/instrumentação , Vetores Genéticos/metabolismo , Humanos , Fígado/virologia , Pulmão/virologia , Camundongos , Filogenia , Baço/virologia , Transdução GenéticaRESUMO
[Figure: see text].
Assuntos
Transição Epitelial-Mesenquimal , Hipertensão Pulmonar/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Camundongos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Longo não Codificante/genética , Transcriptoma , Remodelação VascularRESUMO
RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. OBJECTIVE: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. METHODS AND RESULTS: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. CONCLUSIONS: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling.
Assuntos
Proliferação de Células/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitose/fisiologia , Músculo Liso Vascular/metabolismo , RNA Longo não Codificante/biossíntese , Remodelação Vascular/fisiologia , Ciclo Celular/fisiologia , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Humanos , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Técnicas de Cultura de Órgãos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Veia Safena/citologia , Veia Safena/metabolismoRESUMO
Atherosclerosis is a complex inflammatory pathology underpinning cardiovascular diseases (CVD), which are the leading cause of death worldwide. The interplay between vascular stromal cells and immune cells is fundamental to the progression and outcome of atherosclerotic disease, however, the majority of in vitro studies do not consider the implications of these interactions and predominantly use mono-culture approaches. Here we present a simple and robust methodology involving the co-culture of vascular endothelial (ECs) and smooth muscle cells (SMCs) alongside an inflammatory compartment, in our study containing THP-1 macrophages, for studying these complex interactions. Using this approach, we demonstrate that the interaction between vascular stromal and immune cells produces unique cellular phenotypes and soluble mediator profiles not observed in double-cell 2D cultures. Our results highlight the importance of cellular communication and support the growing idea that in vitro research must evolve from mono-culture systems to provide data more representative of the multi-cellular environment found in vivo. The methodology presented, in comparison with established approaches, has the advantage of being technically simple whilst enabling the isolation of pure populations of ECs, SMCs and immune cells directly from the co-culture without cell sorting. The approach described within would be applicable to those studying mechanisms of vascular inflammation, particularly in relation to understanding the impact cellular interaction has on the cumulative immune-vascular response to atherogenic or inflammatory stimuli.
Assuntos
Aterosclerose/etiologia , Aterosclerose/patologia , Comunicação Celular , Técnicas de Cultura de Células , Técnicas de Cocultura , Modelos Biológicos , Biomarcadores , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Imunofluorescência , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Músculo Liso Vascular , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismoRESUMO
Excessive vascular smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis are key events in the development of intimal hyperplasia, a pathophysiological response to acute or chronic sources of vascular damage that can lead to occlusive narrowing of the vessel lumen. Atherosclerosis, the primary cause of coronary artery disease, is characterised by chronic vascular inflammation and dyslipidemia, while revascularisation surgeries such as coronary stenting and bypass grafting represent acute forms of vascular injury. Gene knockouts of transforming growth factor-beta (TGFß), its receptors and downstream signalling proteins have demonstrated the importance of this pleiotropic cytokine during vasculogenesis and in the maintenance of vascular homeostasis. Dysregulated TGFß signalling is a hallmark of many vascular diseases, and has been associated with the induction of pathological vascular cell phenotypes, fibrosis and ECM remodelling. Here we present an overview of TGFß signalling in SMCs, highlighting the ways in which this multifaceted cytokine regulates SMC behaviour and phenotype in cardiovascular diseases driven by intimal hyperplasia.
Assuntos
Doença da Artéria Coronariana/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Movimento Celular , Proliferação de Células , Doença da Artéria Coronariana/patologia , Humanos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/análiseRESUMO
Vascular immune-inflammatory responses play a crucial role in the progression and outcome of atherosclerosis. The ability to assess localized inflammation through detection of specific vascular inflammatory biomarkers would significantly improve cardiovascular risk assessment and management; however, no multi-parameter molecular imaging technologies have been established to date. Here, we report the targeted in vivo imaging of multiple vascular biomarkers using antibody-functionalized nanoparticles and surface-enhanced Raman scattering (SERS). Methods: A series of antibody-functionalized gold nanoprobes (BFNP) were designed containing unique Raman signals in order to detect intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin using SERS. Results: SERS and BFNP were utilized to detect, discriminate and quantify ICAM-1, VCAM-1 and P-selectin in vitro on human endothelial cells and ex vivo in human coronary arteries. Ultimately, non-invasive multiplex imaging of adhesion molecules in a humanized mouse model was demonstrated in vivo following intravenous injection of the nanoprobes. Conclusion: This study demonstrates that multiplexed SERS-based molecular imaging can indicate the status of vascular inflammation in vivo and gives promise for SERS as a clinical imaging technique for cardiovascular disease in the future.
Assuntos
Vasos Coronários/diagnóstico por imagem , Vasos Coronários/imunologia , Células Endoteliais da Veia Umbilical Humana/química , Imagem Molecular/métodos , Análise Espectral Raman/métodos , Animais , Feminino , Ouro/química , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Imagem Molecular/instrumentação , Nanopartículas/química , Selectina-P/genética , Selectina-P/imunologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Human adenoviral serotype 5 (HAdV-5) vectors have predominantly hepatic tropism when delivered intravascularly, resulting in immune activation and toxicity. Coagulation factor X (FX) binding to HAdV-5 mediates liver transduction and provides protection from virion neutralization in mice. FX is dispensable for liver transduction in mice lacking IgM antibodies or complement, suggesting that alternative transduction pathways exist. To identify novel factor(s) mediating HAdV-5 FX-independent entry, we investigated HAdV-5 transduction in vitro in the presence of serum from immunocompetent C57BL/6 or immunocompromised mice lacking IgM antibodies (Rag 2-/- and NOD-scid-gamma [NSG]). Sera from all three mouse strains enhanced HAdV-5 transduction of A549 cells. While inhibition of HAdV-5-FX interaction with FX-binding protein (X-bp) inhibited transduction in the presence of C57BL/6 serum, it had negligible effect on the enhanced transduction observed in the presence of Rag 2-/- or NSG serum. Rag 2-/- serum also enhanced transduction of the FX binding-deficient HAdV-5HVR5*HVR7*E451Q (AdT*). Interestingly, Rag 2-/- serum enhanced HAdV-5 transduction in a FX-independent manner in CHO-CAR and SKOV3-CAR cells (CHO or SKOV3 cells transfected to stably express human coxsackievirus and adenovirus receptor [CAR]). Additionally, blockade of CAR with soluble HAdV-5 fiber knob inhibited mouse serum-enhanced transduction in A549 cells, suggesting a potential role for CAR. Transduction of HAdV-5 KO1 and HAdV-5/F35 (CAR binding deficient) in the presence of Rag 2-/- serum was equivalent to that of HAdV-5, indicating that direct interaction between HAdV-5 and CAR is not required. These data suggest that FX may protect HAdV-5 from neutralization but has minimal contribution to HAdV-5 transduction in the presence of immunocompromised mouse serum. Alternatively, transduction occurs via an unidentified mouse serum protein capable of bridging HAdV-5 to CAR.IMPORTANCE The intravascular administration of HAdV-5 vectors can result in acute liver toxicity, transaminitis, thrombocytopenia, and injury to the vascular endothelium, illustrating challenges yet to overcome for HAdV-5-mediated systemic gene therapy. The finding that CAR and potentially an unidentified factor present in mouse serum might be important mediators of HAdV-5 transduction highlights that a better understanding of the complex biology defining the interplay between adenovirus immune recognition and cellular uptake mechanisms is still required. These findings are important to inform future optimization and development of HAdV-5-based adenoviral vectors for gene therapy.
Assuntos
Adenovírus Humanos/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Vetores Genéticos , Soro/imunologia , Células A549 , Adenovírus Humanos/classificação , Animais , Linhagem Celular , Linhagem Celular Tumoral , Fator X/metabolismo , Humanos , Imunocompetência , Hospedeiro Imunocomprometido , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Ligação Proteica , Sorogrupo , Tropismo ViralRESUMO
Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells and endothelial cells associated with obliteration of small pulmonary arterioles and formation of plexiform lesions. To date, no curative treatments have been identified for pulmonary arterial hypertension. There are various therapeutic options, including conventional medical therapies and oral, subcutaneous, intravenous, and inhalation delivery. We have previously shown that miR-143/145 knockout can prevent the development of chronic hypoxia-induced pulmonary hypertension (PH) in mice. Here, we use chronic hypoxia-induced PH as a disease model to evaluate miR-143/145 inhibition after delivery of antimiRNAs via the subcutaneous or intranasal routes. We use qRT-PCR and immunofluorescence to confirm that both delivery strategies efficiently inhibit miR-143/145 in lung tissue from mice with chronic hypoxia-induced PH.
Assuntos
Técnicas de Transferência de Genes , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/terapia , Pulmão/metabolismo , MicroRNAs/administração & dosagem , Administração Intranasal , Animais , Antagomirs/administração & dosagem , Carbocianinas/metabolismo , Doença Crônica , Modelos Animais de Doenças , Feminino , Imunofluorescência , Hipertrofia Ventricular Direita/diagnóstico , Hipóxia/complicações , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE OF REVIEW: Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are becoming fundamentally important in the pathophysiology relating to injury-induced vascular remodelling. We highlight recent studies that demonstrate the involvement of ncRNAs in vein graft disease, in in-stent restenosis and in pulmonary arterial hypertension, with a particular focus on endothelial cell and vascular smooth muscle cell function. We also briefly discuss the emerging role of exosomal-derived ncRNAs and how this mechanism impacts on vascular function. RECENT FINDINGS: ncRNAs have been described as novel regulators in the pathophysiology of vascular injury, inflammation, and vessel wall remodelling. In particular, several studies have demonstrated that manipulation of miRNAs can reduce the burden of pathological vascular remodelling. Such studies have also shown that exosomal miRNA-mediated, cell-to-cell communication between endothelial cells and vascular smooth muscle cells is critical in the disease process. In addition to miRNAs, lncRNAs are emerging as regulators of vascular function in health and disease. Although lncRNAs are complex in both their sheer numbers and mechanisms of action, identifying their contribution to vascular disease is essential. SUMMARY: Given the important roles of ncRNAs in vascular injury and remodelling together will their capacity for cell-to-cell communication, manipulating ncRNA might provide novel therapeutic interventions.
Assuntos
RNA não Traduzido/genética , Remodelação Vascular/genética , Animais , Humanos , Transcrição GênicaRESUMO
RATIONALE: The pathogenesis of pulmonary arterial hypertension (PAH) remains unclear. The 4 microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. OBJECTIVE: To elucidate the transcriptional regulation of the miR-143/145 cluster and the role of miR-143 in PAH. METHODS AND RESULTS: We identified the promoter region that regulates miR-143/145 microRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signaling pathways, including estrogen receptor, liver X factor/retinoic X receptor, transforming growth factor-ß (Smads), and hypoxia (hypoxia response element), that regulated levels of all pri-miR stem loop transcription and resulting microRNA expression. We observed that miR-143-3p is selectively upregulated compared with miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMC-derived exosomes. Using assays with pulmonary arterial endothelial cells, we demonstrated a paracrine promigratory and proangiogenic effect of miR-143-3p-enriched exosomes from PASMC. Quantitative polymerase chain reaction and in situ hybridization showed elevated expression of miR-143 in calf models of PAH and in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role of miR-143 in experimental pulmonary hypertension in vivo in miR-143-/- and anti-miR-143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. CONCLUSIONS: MiR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, whereas inhibition of miR-143-3p blocked experimental pulmonary hypertension. Taken together, these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.
Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Hipertensão Pulmonar/metabolismo , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Pressão Arterial , Sítios de Ligação , Estudos de Casos e Controles , Bovinos , Movimento Celular , Células Endoteliais/patologia , Exossomos/metabolismo , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Regiões Promotoras Genéticas , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Remodelação Vascular , Função Ventricular Direita , Pressão VentricularRESUMO
Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein.
Assuntos
Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Vetores Genéticos/genética , Adenovírus Humanos/metabolismo , Sítios de Ligação , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Vetores Genéticos/metabolismo , Vetores Genéticos/normas , Humanos , Integrinas/metabolismo , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmídeos/normas , Ligação Proteica , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. METHODS: Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). RESULTS: Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. CONCLUSIONS: We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Capsídeo , Células Cultivadas , Humanos , Veia Safena , Inibidor Tecidual de Metaloproteinase-3/análise , Inibidor Tecidual de Metaloproteinase-3/farmacologia , Transdução Genética , beta-Galactosidase/análiseRESUMO
Cardiovascular disease is the most frequent cause of mortality in the western world, accounting for over 800,000 premature deaths per year in the EU alone. Cardiovascular disease is the second most common application for gene therapy clinical trials, which most frequently employ adenovirus serotype 5 (Ad5)-based vectors as delivery vehicles. Although interactions of Ad5 vectors with circulating proteins and cells can limit their efficacy after systemic administration, local gene delivery strategies show great potential in the cardiovascular setting, notably in the context of vascular delivery. Here we review the pathogenesis of bypass graft failure and in-stent restenosis, identifying potential therapeutic targets and discussing recent advances in the field of adenovirus biology and retargeting that, in concert, will potentially translate in coming years to more effective gene therapies for cardiovascular applications.
Assuntos
Doenças Cardiovasculares/terapia , Ponte de Artéria Coronária/métodos , Terapia Genética/métodos , Adenoviridae/genética , Animais , Doenças Cardiovasculares/fisiopatologia , Reestenose Coronária/patologia , Vetores Genéticos , Humanos , Terapia de Alvo Molecular , Falha de Prótese , StentsRESUMO
The development of adenoviral vectors for intravascular (i.v.) delivery will require improvements to their in vivo safety and efficacy. The hypervariable regions (HVRs) of the Ad5 hexon are a target for neutralizing antibodies, but also interact with factor X (FX), facilitating hepatocyte transduction. Ad48, a species D adenovirus, does not bind FX and has low seroprevalence. Therefore, it has been suggested that Ad5HVR48(1-7), a hexon-chimeric vector featuring the seven HVRs from Ad48, should display advantageous properties for gene therapy, by evading pre-existing Ad5 immunity and blocking FX interactions. We investigated the in vivo biodistribution of Ad5, Ad5HVR48(1-7), and Ad48 following i.v. delivery. Ad5HVR48(1-7) displayed reduced hepatocyte transduction and accumulation in Kupffer cells (KCs), but triggered a robust proinflammatory response, even at relatively low doses of vector. We detected elevated serum transaminases (48 hours) and increased numbers of periportal CD11b(+)/Gr-1(+) cells in the livers of Ad5HVR48(1-7)-treated animals following i.v., but not intramuscular (i.m.), delivery. In contrast, Ad48 did not elevate transaminases or result in the accumulation of CD11b(+)/Gr-1(+) cells. Collectively, these findings suggest that substantial hexon modifications can lead to unexpected properties which cannot be predicted from parental viruses. Therefore, refined mutations may be preferential for the successful development of targeted vector systems which require i.v. administration.
Assuntos
Adenoviridae/imunologia , Administração Intravenosa , Vetores Genéticos/efeitos adversos , Vetores Genéticos/imunologia , Animais , Vetores Genéticos/administração & dosagem , Células Hep G2 , Humanos , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Transaminases/genética , Transaminases/metabolismoRESUMO
The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton-α(v)integrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1+ sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular delivery.
Assuntos
Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Vetores Genéticos , Mutagênese Sítio-Dirigida , Mutação , Adenovírus Humanos/química , Adenovírus Humanos/metabolismo , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Citocinas/imunologia , Endocitose , Fator X/química , Fator X/metabolismo , Humanos , Imunidade Inata , Imuno-Histoquímica , Fígado/imunologia , Fígado/metabolismo , Fígado/virologia , Camundongos , Camundongos Endogâmicos , Microscopia de Fluorescência , Oligopeptídeos/metabolismo , Ligação Proteica , Sorotipagem , Baço/imunologia , Baço/metabolismo , Baço/virologia , Distribuição Tecidual , Transdução Genética , beta-Galactosidase/genéticaRESUMO
Nanomedicine based on the use of adenovirus vectors for therapeutic gene delivery shows broad potential. Specific targeting for many gene therapy applications, such as metastatic cancers or cardiovascular diseases requires intravascular delivery of the vector. However, a major barrier to successful adenovirus vector targeting follows systemic delivery, as upon contact with the bloodstream the virus interacts with a variety of host proteins, in particular coagulation factor X, which mediates profound liver gene transfer. This inherent hepatic tropism combined with macrophage scavenging minimizes the efficacy of the virus at the desired sites and induces toxic side effects. Understanding the complex, multifaceted interactions of adenovirus with host factors is of vital importance to the design of safer vectors with improved efficacy and pharmacokinetic profiles. Increased knowledge of adenovirus biology provides the opportunity to develop innovative strategies to detarget the virus from the liver following intravascular delivery and redirect the vector to disease areas.
Assuntos
Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Transfecção/métodosRESUMO
Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex.
Assuntos
Adenoviridae/metabolismo , Aminoácidos Básicos/metabolismo , Fator X/metabolismo , Substituição de Aminoácidos/genética , Aminoácidos Básicos/genética , Fator X/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or α(v) integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for α(v) integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of α(v) integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define organ targeting following contact of human adenoviruses with blood.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/fisiologia , Fator X/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Células Hep G2 , Proteoglicanas de Heparan Sulfato/metabolismo , Proteoglicanas de Heparan Sulfato/fisiologia , Heparina/farmacologia , Humanos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Oligopeptídeos/química , Oligopeptídeos/fisiologia , Organismos Geneticamente Modificados , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/fisiologia , Sulfatos/metabolismo , Células Tumorais Cultivadas , Internalização do Vírus/efeitos dos fármacosRESUMO
A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c(+), ER-TR7(+), and MAdCAM-1(+) cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46(+) mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.
Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Fator X/metabolismo , Vetores Genéticos , Adenovírus Humanos/classificação , Animais , Proteínas do Capsídeo/genética , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Fígado/metabolismo , Fígado/virologia , Pulmão/metabolismo , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sorotipagem , Baço/metabolismo , Baço/virologia , Fatores de Tempo , Distribuição Tecidual , Transdução Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and "bridging" interactions. "Bridging" interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of "bridging interactions" such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated "stealth" vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.