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Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics.
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The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.
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Epilepsias Mioclônicas , Canal de Sódio Disparado por Voltagem NAV1.1 , Animais , Camundongos , Modelos Animais de Doenças , DNA Complementar , Epilepsias Mioclônicas/terapia , Epilepsias Mioclônicas/tratamento farmacológico , Neurônios GABAérgicos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , FenótipoRESUMO
BACKGROUND AND AIMS: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. METHODS AND RESULTS: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. CONCLUSIONS: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production.
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Adenoviridae , Integrases , Adenoviridae/genética , Integrases/genética , Vetores Genéticos/genética , Proteínas Virais/genéticaRESUMO
Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27-hydroxylase. Disruption of the bile acid biosynthesis pathway and accumulation of toxic precursors such as cholestanol cause chronic diarrhea, bilateral juvenile cataracts, tissue deposition of cholestanol and cholesterol (xanthomas), and progressive motor/neuropsychiatric alterations. We have evaluated the therapeutic potential of adeno-associated virus (AAV) vectors expressing CYP27A1 in a CTX mouse model. We found that a vector equipped with a strong liver-specific promoter (albumin enhancer fused with the α1 anti-trypsin promoter) is well tolerated and shows therapeutic effect at relatively low doses (1.5 × 1012 viral genomes [vg]/kg), when less than 20% of hepatocytes overexpress the transgene. This vector restored bile acid metabolism and normalized the concentration of most bile acids in plasma. By contrast, standard treatment (oral chenodeoxycholic acid [CDCA]), while reducing cholestanol, did not normalize bile acid composition in plasma and resulted in supra-physiological levels of CDCA and its derivatives. At the transcriptional level, only the vector was able to avoid the induction of xenobiotic-induced pathways in mouse liver. In conclusion, the overexpression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in Cyp27a1 -/- mice. These features make gene therapy a feasible option for the etiological treatment of CTX patients.
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Dravet syndrome is a genetic encephalopathy characterized by severe epilepsy combined with motor, cognitive, and behavioral abnormalities. Current antiepileptic drugs achieve only partial control of seizures and provide little benefit on the patient's neurological development. In >80% of cases, the disease is caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of the Nav1.1 voltage-gated sodium channel. Novel therapies aim to restore SCN1A expression in order to address all disease manifestations. We provide evidence that a high-capacity adenoviral vector harboring the 6-kb SCN1A cDNA is feasible and able to express functional Nav1.1 in neurons. In vivo, the best biodistribution was observed after intracerebral injection in basal ganglia, cerebellum, and prefrontal cortex. SCN1A A1783V knockin mice received the vector at 5 weeks of age, when most neurological alterations were present. Animals were protected from sudden death, and the epileptic phenotype was attenuated. Improvement of motor performance and interaction with the environment was observed. In contrast, hyperactivity persisted, and the impact on cognitive tests was variable (success in novel object recognition and failure in Morris water maze tests). These results provide proof of concept for gene supplementation in Dravet syndrome and indicate new directions for improvement.
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Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.
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Anticorpos Bloqueadores/genética , Antígeno B7-H1/metabolismo , Carcinoma/terapia , Terapia Genética/métodos , Imunoterapia/métodos , Macrófagos/imunologia , Neoplasias Peritoneais/terapia , Adenoviridae/genética , Animais , Anticorpos Bloqueadores/imunologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linhagem Celular , Feminino , Vetores Genéticos/genética , Inibidores de Checkpoint Imunológico/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Fatores Imunológicos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Poli I-C/uso terapêuticoRESUMO
Cytokines are small proteins that are crucial for controlling the growth and activity of blood cells and other cells of the immune system [...].
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Osteosarcoma is the most frequent and aggressive bone tumor in children and adolescents, with a long-term survival rate of 30%. Interleukin-12 (IL-12) is a potent cytokine that bridges innate and adaptive immunity, triggers antiangiogenic responses, and achieves potent antitumor effects. In this work, we evaluated the antisarcoma effect of a high-capacity adenoviral vector encoding mouse IL-12. This vector harbored a mifepristone-inducible system for controlled expression of IL-12 (High-Capacity adenoviral vector enconding the EF1α promoter [HCA-EFZP]-IL-12). We found that local administration of the vector resulted in a reduction in the tumor burden, extended overall survival, and tumor eradication. Moreover, long-term survivors exhibited immunological memory when rechallenged with the same tumor cells. Treatment with HCA-EFZP-IL-12 also resulted in a significant decrease in lung metastasis. Immunohistochemical analyses showed profound remodeling of the osteosarcoma microenvironment with decreases in angiogenesis and macrophage and myeloid cell numbers. In summary, our data underscore the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which can trigger a potent antitumor immune response in primary and metastatic pediatric osteosarcoma.
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Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrA was addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrA wt-treated mice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-αR (IFNAR)-/- animals were additionally treated with anti-TNF-α (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR-/- and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-α-treated IFNAR-/- animals. No effect of AdrA wt was seen in STING-deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-α are both required and act synergistically.
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Células Dendríticas/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Proteínas de Membrana/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Adenoviridae/genética , Animais , Antivirais/uso terapêutico , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Imunomodulação , Interferon Tipo I/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptores Adrenérgicos alfa 1/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação ViralRESUMO
The adaptation of adenoviruses as gene delivery tools has resulted in the development of high-capacity adenoviral vectors (HC-AdVs), also known, helper-dependent or "gutless". Compared with earlier generations (E1/E3-deleted vectors), HC-AdVs retain relevant features such as genetic stability, remarkable efficacy of in vivo transduction, and production at high titers. More importantly, the lack of viral coding sequences in the genomes of HC-AdVs extends the cloning capacity up to 37 Kb, and allows long-term episomal persistence of transgenes in non-dividing cells. These properties open a wide repertoire of therapeutic opportunities in the fields of gene supplementation and gene correction, which have been explored at the preclinical level over the past two decades. During this time, production methods have been optimized to obtain the yield, purity, and reliability required for clinical implementation. Better understanding of inflammatory responses and the implementation of methods to control them have increased the safety of these vectors. We will review the most significant achievements that are turning an interesting research tool into a sound vector platform, which could contribute to overcome current limitations in the gene therapy field.
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Adenoviridae/genética , Tratamento Farmacológico/métodos , Vetores Genéticos/genética , Adenoviridae/imunologia , Animais , Vetores Genéticos/efeitos adversos , Vetores Genéticos/normas , Instabilidade Genômica , HumanosRESUMO
IL-8 (CXCL-8) is a chemoattractant factor for myeloid leukocytes, that is produced in large quantities by many solid tumors. Levels of IL-8, which can act upon a variety of immune and nonimmune cells, can tell us a lot about tumors, including their size (positive association) and how likely they are to respond to immunotherapy (negative association). This is because the IL-8 produced by tumors can promote angiogenesis, recruit immunosuppressive cells like neutrophils and myeloid-derived suppressor cells (MDSCs), and stimulate epithelial-to-mesenchymal transition, which is a precursor to metastasis. In a pooled analysis of several clinical trials in kidney cancer, melanoma, and lung cancer, it was found that patients with higher baseline concentrations of IL-8 in the blood experienced worse outcomes and lower overall survival after being treated with immunotherapy. Currently, the field that relates IL-8 to immunotherapy is leading to numerous and promising clinical trials that combine the inhibition of IL-8 with existing immunotherapeutic therapies. For this reason, multiple constructs based on IL-8 agonists are being developed clinically by the pharmaceutical and biotech industries.
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Melanoma , Células Supressoras Mieloides , Humanos , Imunoterapia , Interleucina-8RESUMO
Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.
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Adenoviridae/genética , Vetores Genéticos/metabolismo , Peixe-Zebra/genética , Animais , Encéfalo/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Modelos Animais , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In this review, we will highlight several studies that revolve around interleukin-8 (IL-8) and show the multiple facets that could take in the tumor microenvironment. Chemokines that attract neutrophils (to a large extent, IL-8) can have a bimodal behavior inducing the migration of them in the first place and later favoring the formation of NETs in the place of emission focus of the chemokine. Also, this mechanism occurs when neutrophils migrate to tumor cells and where the extrusion of NETs in the tumor is observed. A possible participation of NETs in cancer progression was considered; however, until now, it is difficult to decide if NETosis plays a pro- or antitumor role, although it is necessary to emphasize that there is more experimentation focused on the protumorigenic aspect of the NETs. The formation of NETs has a relevant role in the inhibition of the immune response against the tumor generated by neutrophils and in turn favoring the processes involved in the development of tumor metastasis. It is striking that we do not have more complete information about the effects of circulating chemokines on neutrophils in cancer patients and hence the suitability of this review. No one has observed to date the impact that it could have on other cell populations to inhibit the arrival of neutrophils and the formation/elimination of NETs. However, the extent to which NETs affect the function of other cells of the immune system in the tumor context has not been directly demonstrated. It is necessary to identify possible combinations of immunotherapy that involve the modulation of neutrophil activity with other strategies (immunomodulatory antibodies or adoptive cell therapy). Therefore, knowing the mechanisms by which tumors take advantage of this ability of neutrophils to form NETs is very important in the search for antitumor therapies and thus be able to take advantage of the possible immunotherapeutic combinations that we currently have in clinical practice.
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Armadilhas Extracelulares/imunologia , Interleucina-8/imunologia , Neoplasias/patologia , Neutrófilos/imunologia , Microambiente Tumoral/imunologia , Animais , Humanos , Camundongos , Neoplasias/imunologia , Ativação de NeutrófiloRESUMO
The repurposing of drugs approved by the regulatory agencies for other indications is emerging as a valuable alternative for the development of new antimicrobial therapies, involving lower risks and costs than the de novo development of novel antimicrobial drugs. Adenovirus infections have showed a steady increment in recent years, with a high clinical impact in both immunosuppressed and immunocompetent patients. In this context, the lack of a specific drug to treat these infections supports the search for new therapeutic alternatives. In this study, we examined the anti-HAdV properties of mifepristone, a commercially available synthetic steroid drug. Mifepristone showed significant in vitro anti-HAdV activity at low micromolar concentrations with little cytotoxicity. Our mechanistic assays suggest that this drug could affect the microtubule transport, interfering with the entry of the virus into the nucleus and therefore inhibiting HAdV infection.
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Infecções por Adenoviridae/tratamento farmacológico , Adenovírus Humanos/efeitos dos fármacos , Antivirais/uso terapêutico , Reposicionamento de Medicamentos , Mifepristona/uso terapêutico , Células A549 , Animais , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Biological therapies based on recombinant proteins such as antibodies or cytokines are continuously improving the repertoire of treatments against cancer. However, safety and efficacy of this approach is often limited by inappropriate biodistribution and pharmacokinetics of the proteins when they are administered systemically. Local administration of gene therapy vectors encoding these proteins would be a feasible alternative if they could mediate long-term and controlled expression of the transgene after a single intratumoral administration. We describe a new vector platform specially designed for this purpose. Different combinations of transactivators and promoters were evaluated to obtain a fully humanized inducible system responsive to the well-characterized drug mifepristone. The optimal transactivator conformation was based on DNA binding domains from the chimeric protein ZFHD1 fused to the progesterone receptor ligand binding domain and the NFkb p65 activation domain. The expression of this hybrid transactivator under the control of the elongation factor 1α (EF1α) or the chimeric CAG promoters ensured functionality of the system in a variety of cancer types. Expression cassettes with luciferase as a reporter gene were incorporated into High-Capacity adenoviral vectors (HC-Ad) for in vivo evaluation. Systemic administration of the vectors into C57BL/6 mice revealed that the vector based on the EF1α promoter (HCA-EF-ZP) allows tight control of transgene expression and remains stable for at least two months, whereas the CAG promoter suffers a progressive inactivation. Using an orthotopic pancreatic cancer model in syngeneic C57BL/6 mice we show that the local administration of HCA-EF-ZP achieves better tumor/liver ratio of luciferase production than the intravenous route. However, regional spread of the vector led to substantial transgene expression in peritoneal organs. We reduced this leakage through genetic modification of the vector capsid to display RGD and poly-lysine motifs in the fiber knob. Safety and antitumor effect of this gene therapy platform was demonstrated using interleukin-12 as a therapeutic gene. In conclusion, we have developed a new tool that allows local, sustained and controlled production of therapeutic proteins in tumors.
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Interleucina-12/genética , Mifepristona/administração & dosagem , Neoplasias Pancreáticas/genética , Transgenes , Microambiente Tumoral/genética , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Feminino , Vetores Genéticos , Humanos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão , Fatores de TranscriçãoRESUMO
BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.
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Reação de Fase Aguda/genética , Dano ao DNA/genética , Estresse do Retículo Endoplasmático/genética , Regeneração Hepática/genética , Fígado/metabolismo , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Células Hep G2 , Hepatectomia , Humanos , Interleucina-6/genética , Camundongos , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND: The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. METHODS: We have developed a protocol consisting of sequential intratumor administrations of new Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the immunostimulatory cytokine oncostatin M (OSM). Transgene expression, toxicity and antitumor effect were evaluated using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, which are sensitive to OSM and permissive for replication of both OVs. RESULTS: NDV-OSM was more cytolytic, whereas Ad-OSM caused higher OSM expression in vivo. Both viruses achieved only a marginal antitumor effect in monotherapy. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe, showed a significant antitumor effect and stimulated immune responses against cancer cells. Similar efficacy was observed when the order of virus administrations was reversed. CONCLUSION: Sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.
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Terapia Viral Oncolítica/métodos , Oncostatina M/biossíntese , Neoplasias Pancreáticas/terapia , Animais , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Cricetinae , Humanos , Mesocricetus , Transplante de Neoplasias , Vírus Oncolíticos/genética , Oncostatina M/genética , Replicação ViralRESUMO
Acute intermittent porphyria (AIP) is a hepatic metabolic disease that results from haplo-insufficient activity of porphobilinogen deaminase (PBGD). The dominant clinical feature is acute intermittent attacks when hepatic heme synthesis is activated by endocrine or exogenous factors. Gene therapy vectors over-expressing PBGD protein in the liver offers potential as a cure for AIP. Here, we developed a helper-dependent adenovirus (HDA) encoding human PBGD (hPBGD) and assessed its therapeutic efficacy in a murine model of AIP. Intravenous or intrahepatic administration of HDA-hPBGD to AIP mice resulted in a sustained hepatic hPBGD expression in a dose-dependent manner. Intrahepatic administration conveyed full protection against induced porphyria attacks at a significantly lower viral dose than intravenous injection. Transgenic hPBGD accumulated only in the cytosol of hepatocytes as the endogenous protein. Characterization of PBGD-deficient mouse strains revealed that a strong PBGD deficiency causes the chronic disturbance of cytosolic and endoplasmic reticulum folding machineries. This disturbance was completely restored over time by the over-expression of hPBGD. HDA-hPBGD is a promising vector that protects against porphyria attacks and resolves the chronic folding stress associated with low levels of PBGD activity.
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Adenoviridae/genética , Terapia Genética , Hidroximetilbilano Sintase/genética , Porfiria Aguda Intermitente/genética , Porfiria Aguda Intermitente/terapia , Adenoviridae/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Hepatócitos/enzimologia , Hepatócitos/virologia , Humanos , Hidroximetilbilano Sintase/metabolismo , Fígado/enzimologia , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Porfiria Aguda Intermitente/enzimologia , Porfiria Aguda Intermitente/prevenção & controle , Dobramento de ProteínaRESUMO
UNLABELLED: Regulatory T cells (Treg) play a critical role in the modulation of immune responses to viral antigens in chronic viral hepatitis. Woodchucks (Marmota monax) infected with the woodchuck hepatitis virus (WHV) represent the best animal model for chronic hepatitis B virus (HBV) infection. Examination of intrahepatic and peripheral Treg in uninfected and WHV chronically infected woodchucks showed a significant increase of intrahepatic Treg numbers in chronically infected animals, whereas no differences were found in peripheral blood. In agreement with these data, higher expression levels of Forkhead box P3 (Foxp3), interleukin (IL)-10, transforming growth factor beta (TGF-ß) were detected in the liver of chronic WHV carriers in comparison to uninfected animals. Furthermore, treatment of WHV-infected animals with an adenovirus encoding IL-12 failed to reduce viral load, a finding that was associated with lymphocyte unresponsiveness to IL-12 stimulation in vitro. We observed that TGF-ß and Treg play a major role in the lack of lymphocyte response to IL-12 stimulation, as TGF-ß inhibition and Treg depletion allowed recovery of T-cell responsiveness to this cytokine. Based on these results, woodchucks were treated with IL-12 in combination with a TGF-ß inhibitory peptide or Treg depletion. However, no antiviral effect was achieved and, instead, an enhancement of the intrahepatic tolerogenic environment was observed. CONCLUSION: Our data show that TGF-ß inhibition or Treg depletion had no added benefit over IL-12 therapy in chronic WHV infection. IL-12 immunostimulation induces a strong immunosuppressive reaction in the liver of chronic WHV carriers that counteracts the antiviral effect of the treatment.
Assuntos
Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Tolerância Imunológica/efeitos dos fármacos , Interleucina-12/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Antígenos Virais/imunologia , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Ciclofosfamida/farmacologia , Quimioterapia Combinada , Vírus da Hepatite B da Marmota/imunologia , Hepatite B Crônica/imunologia , Tolerância Imunológica/imunologia , Imunossupressores/farmacologia , Interleucina-12/imunologia , Neoplasias Hepáticas , Marmota , Peptídeos/imunologia , Peptídeos/farmacologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologia , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 µg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine. CONCLUSIONS: Long-term controlled expression of IL-12 using an HC-Ad vector in combination with oxaliplatin is effective and clinically applicable against hepatic colon cancer metastases.