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Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.
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BACKGROUND: The Spike protein mutation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to decreased protective effect of various vaccines and mAbs, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin-converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Downregulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed on the basis of sequence data, require no delivery system, and can be administered locally. OBJECTIVE: We sought to design ASOs that can block SARS-CoV-2 by downregulating ACE2 in human airway. METHODS: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASO-pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. RESULTS: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore, they efficiently suppressed virus replication of 3 different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, ASOs also downregulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathologic changes in lungs, and increased survival of mice. CONCLUSIONS: ACE2-targeting ASOs can effectively block SARS-CoV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high-risk populations.
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Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-ß/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-ß driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-ß response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.
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Fibrose Pulmonar Idiopática , Fatores de Transcrição , Camundongos , Animais , Humanos , Proliferação de Células , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Homeodomínio/genética , Proteína Fosfatase 2CRESUMO
BACKGROUND: Although immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may be due to a poor pre-existing network linking innate and adaptive immunity. Here, we present an antisense oligonucleotide (ASO)-based strategy that dually targets toll-like receptor 9 (TLR9) and programmed cell death ligand 1 (PD-L1), aiming to overcome resistance to anti-PD-L1 monoclonal therapy. METHODS: We designed a high-affinity immunomodulatory IM-TLR9:PD-L1-ASO antisense oligonucleotide (hereafter, IM-T9P1-ASO) targeting mouse PD-L1 messenger RNA and activating TLR9. Then, we performed in vitro and in vivo studies to validate the IM-T9P1-ASO activity, efficacy, and biological effects in tumors and draining lymph nodes. We also performed intravital imaging to study IM-T9P1-ASO pharmacokinetics in the tumor. RESULTS: IM-T9P1-ASO therapy, unlike PD-L1 antibody therapy, results in durable antitumor responses in multiple mouse cancer models. Mechanistically, IM-T9P1-ASO activates a state of tumor-associated dendritic cells (DCs), referred to here as DC3s, which have potent antitumor potential but express the PD-L1 checkpoint. IM-T9P1-ASO has two roles: it triggers the expansion of DC3s by engaging with TLR9 and downregulates PD-L1, thereby unleashing the antitumor functions of DC3s. This dual action leads to tumor rejection by T cells. The antitumor efficacy of IM-T9P1-ASO depends on the antitumor cytokine interleukin-12 (IL-12), produced by DC3s, and Batf3, a transcription factor required for DC development. CONCLUSIONS: By simultaneously targeting TLR9 and PD-L1, IM-T9P1-ASO amplifies antitumor responses via DC activation, leading to sustained therapeutic efficacy in mice. By highlighting differences and similarities between mouse and human DCs, this study could serve to develop similar therapeutic strategies for patients with cancer.
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Neoplasias , Receptor Toll-Like 9 , Humanos , Camundongos , Animais , Receptor Toll-Like 9/metabolismo , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Oligonucleotídeos Antissenso , Células DendríticasRESUMO
Antisense oligonucleotides (ASOs) are a novel therapeutic strategy that targets a specific gene and suppresses its expression. The cryopyrin-associated periodic syndromes (CAPS) are a spectrum of autoinflammatory diseases characterized by systemic and tissue inflammation that is caused by heterozygous gain-of-function mutations in the nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) gene. The aim of this study was to investigate the efficacy of an Nlrp3-specific ASO treatment in CAPS. An Nlrp3-specific ASO was designed and tested in murine cell lines and bone marrow-derived macrophages (BMDMs) from wild-type and CAPS mouse models. Nlrp3 knock-in mice were treated in vivo with Nlrp3-specific ASO, survival was monitored, and expression of organ-specific Nlrp3 and IL-1ß was measured. Nlrp3-specific ASO treatment of murine cell lines and BMDMs showed a significant downregulation of Nlrp3 and mature IL-1ß protein expression. Ex vivo treatment of Nlrp3 mutant mouse-derived BMDMs with Nlrp3-specific ASO demonstrated significantly reduced IL-1ß release. In vivo, Nlrp3-specific ASO treatment of Nlrp3 mutant mice prolonged survival, reduced systemic inflammation, and decreased tissue-specific expression of Nlrp3 and mature IL-1ß protein. The results of this study demonstrate that Nlrp3-specific ASO treatment downregulates Nlrp3 expression and IL-1ß release in CAPS models, suggesting ASO therapy as a potential treatment of CAPS and other NLRP3-mediated diseases.
Assuntos
Síndromes Periódicas Associadas à Criopirina , Proteína 3 que Contém Domínio de Pirina da Família NLR , Camundongos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Síndromes Periódicas Associadas à Criopirina/genética , Inflamação , Proteínas de Transporte/genética , Interleucina-1beta/metabolismoRESUMO
Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.
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Glucose , Linfadenopatia , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , TriglicerídeosRESUMO
BACKGROUND: Maladaptive endoplasmic reticulum stress signaling in diabetic kidney disease (DKD) is linked to increased glomerular and tubular expression of the cell-death-promoting transcription factor C/EBP homologous protein (CHOP). Here, we determined whether locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) targeting CHOP ameliorate experimental DKD. METHODS: We determined the efficacy of CHOP-ASO in the early and late stages of experimental DKD (in 8- or 16-week-old db/db mice, respectively) alone or with an angiotensin-converting enzyme inhibitor (ACEi), after an in vivo dose-escalation study. We used renal functional parameters and morphologic analyses to assess the effect of CHOP-ASO and renal gene-expression profiling to identify differentially regulated genes and pathways. Several human CHOP-ASOs were tested in hyperglycemia-exposed human kidney cells. RESULTS: CHOP-ASOs efficiently reduced renal CHOP expression in diabetic mice and reduced markers of DKD at the early and late stages. Early combined intervention (CHOP-ASO and ACEi) efficiently prevented interstitial damage. At the later timepoint, the combined treatment reduced indices of both glomerular and tubular damage more efficiently than either intervention alone. CHOP-ASO affected a significantly larger number of genes and disease pathways, including reduced sodium-glucose transport protein 2 (Slc5a2) and PROM1 (CD133). Human CHOP-ASOs efficiently reduced glucose-induced CHOP and prevented death of human kidney cells in vitro . CONCLUSIONS: The ASO-based approach efficiently reduced renal CHOP expression in a diabetic mouse model, providing an additional benefit to an ACEi, particularly at later timepoints. These studies demonstrate that ASO-based therapies efficiently reduce maladaptive CHOP expression and ameliorate experimental DKD.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Camundongos , Humanos , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/prevenção & controle , Diabetes Mellitus Experimental/complicações , Glomérulos Renais , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Rim , Oligonucleotídeos Antissenso/farmacologiaRESUMO
Locked nucleic acid-modified antisense oligonucleotides (ASOs) can achieve strongly different degrees of target knockdown despite having similar biophysical properties and 100% homology with their target. The determinants for this observation remain largely unknown. We used multi-specific ASOs that have 100% sequence complementarity with a common target (IDO1) and a different number of diverse targets and investigated their effect on gene expression in a cell line by RNA-sequencing. We observed a significant higher chance for downregulation of long genes compared to short genes, of genes with high compared to lower expression, and of genes that have more than one binding site for the respective ASO. By investigating the expression of genes that have binding sites for more than one ASO we identified the individual binding site being an important determinant for activity. Under the selected experimental conditions we have not seen indications that availability of RNase H is a limiting factor as the number of degraded target RNA molecules correlated significantly with the number of predicted target RNA molecules. Taken together, by using multi-specific ASOs as tool compounds we identified determinants for ASO activity that can be taken into consideration to improve the selection process of highly potent and selective ASOs in the future.
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Oligonucleotídeos Antissenso , Ribonuclease H , Sítios de Ligação , Linhagem Celular , Oligonucleotídeos Antissenso/genética , RNA , Ribonuclease H/genéticaRESUMO
BACKGROUND: The field of antisense oligonucleotide therapeutics is rapidly growing and in addition to small molecules and therapeutic antibodies, oligonucleotide-based gene expression modifiers have been developed as fully accepted therapeutics. Antisense oligonucleotides are designed to modify gene expression of their specific target genes. However, as their effect relies on Watson-Crick base pairing, they could also bind to other unintended complementary RNAs showing sufficient sequence homology, which in turn could lead to off-target effects. It is assumed that these off-target effects depend on the degree of complementarity between the antisense oligonucleotides and off-target sequences. OBJECTIVE: Aim of this study was the investigation of the effects of antisense oligonucleotides on the expression of potential off-targets having a defined number of mismatches to the oligonucleotide sequence. METHODS: We extend recent studies by investigating the off-target profile of two 17-mer antisense oligonucleotides in two distinct human cell lines by a whole-transcriptome study using RNA sequencing. RESULTS: The relatively high percentage of significantly downregulated off-target genes for which one mismatch is present corroborates the requirement for intense bioinformatic screens and stringent specificity criteria to design antisense oligonucleotides with only minimal sequence complementarity to any non-target sequence. CONCLUSIONS: Avoiding suppression of off-target genes by a thorough bioinformatics screen should strongly reduce the risk for toxicities caused by antisense oligonucleotide-mediated off-target RNA suppression and finally result in safer antisense oligonucleotide-based therapeutics.
Assuntos
Perfilação da Expressão Gênica/métodos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Oligonucleotídeos Antissenso/genética , Pareamento de Bases , Sequência de Bases , Linhagem Celular , Regulação para Baixo , Humanos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Análise de Sequência de RNA , Homologia de Sequência do Ácido NucleicoRESUMO
Colorectal and lung cancers account for one-third of all cancer-related deaths worldwide. Previous studies suggested that metadherin (MTDH) is involved in the development of colorectal and lung cancers. However, how MTDH regulates the pathogenesis of these cancers remains largely unknown. Using genetically modified mouse models of spontaneous colorectal and lung cancers, we found that MTDH promotes cancer progression by facilitating Wnt activation and by inducing cytotoxic T-cell exhaustion, respectively. Moreover, we developed locked nucleic acid-modified (LNA) MTDH antisense oligonucleotides (ASO) that effectively and specifically suppress MTDH expression in vitro and in vivo. Treatments with MTDH ASOs in mouse models significantly attenuated progression and metastasis of colorectal, lung, and breast cancers. Our study opens a new avenue for developing therapies against colorectal and lung cancers by targeting MTDH using LNA-modified ASO. SIGNIFICANCE: This study provides new insights into the mechanism of MTDH in promoting colorectal and lung cancers, as well as genetic and pharmacologic evidence supporting the development of MTDH-targeting therapeutics.
Assuntos
Adenocarcinoma/terapia , Neoplasias Colorretais/terapia , Neoplasias Pulmonares/terapia , Proteínas de Membrana/antagonistas & inibidores , Oligonucleotídeos Antissenso/uso terapêutico , Proteínas de Ligação a RNA/antagonistas & inibidores , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Metástase Neoplásica , Oligonucleotídeos/farmacologia , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Proteínas de Ligação a RNA/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The adenosine axis contributes to the suppression of antitumor immune responses. The ectonucleotidase CD39 degrades extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is degraded to adenosine by CD73. Adenosine binds to, e.g., the A2a receptor (A2aR), which reportedly suppresses effector immune cells. We investigated effects of ATP, AMP, and adenosine analogs on T cell proliferation, apoptosis, and proinflammatory cytokine secretion. CD39 and CD73 expression were suppressed using antisense oligonucleotides (ASOs), and A2aR was blocked using small molecules. Addition of ATP to T cells reduced proliferation and induced apoptosis. Intriguingly, those effects were reverted by suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogs did not suppress proliferation but inhibited secretion of proinflammatory cytokines. Here, we suggest that suppression of T cell proliferation is not directly mediated by A2aR but by intracellular downstream metabolites of adenosine, as blockade of the equilibrative nucleoside transporter (ENT) or adenosine kinase rescued proliferation and prevented induction of apoptosis. In conclusion, adenosine might primarily affect cytokine secretion directly via adenosine receptors, whereas adenosine metabolites might impair T cell proliferation and induce apoptosis. Therefore, inhibition of CD39 and/or CD73 has evident advantages over A2aR blockade to fully revert suppression of antitumor immune responses by the adenosine axis.
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Tumors can utilize a diverse repertoire of immunosuppressive mechanisms to evade attack by the immune system. Despite promising success with blockade of immune checkpoints like PD-1 the majority of patients does not respond to current immunotherapies. The degradation of tryptophan into immunosuppressive kynurenine is an important immunosuppressive pathway. Recent attempts to target the key enzymes of this pathway-IDO1 and TDO2-have so far failed to show therapeutic benefit in the clinic, potentially caused by insufficient target engagement. We, therefore, sought to add an alternative, highly efficient approach to block the degradation of tryptophan by inhibiting the expression of IDO1 and TDO2 using locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs). We show that LNA-modified ASOs can profoundly inhibit the expression of IDO1 and TDO2 in cancer cells in vitro without using a transfection reagent with IC50 values in the sub-micromolar range. We furthermore measured kynurenine production by ASO-treated cancer cells in vitro and observed potently reduced kynurenine levels. Accordingly, inhibiting IDO1 expression in cancer cells in an in vitro system leads to increased proliferation of activated T cells in coculture. We furthermore show that combined treatment of cancer cells in vitro with IDO1-specific ASOs and small molecule inhibitors can reduce the production of kynurenine by cancer cells in a synergistic manner. In conclusion, we propose that a combination of LNA-modified ASOs and small molecule inhibitors should be considered as a strategy for efficient blockade of the degradation of tryptophan into kynurenine in cancer immunotherapy.
Assuntos
Antineoplásicos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Neoplasias/terapia , Oligonucleotídeos Antissenso/farmacologia , Triptofano Oxigenase/antagonistas & inibidores , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Concentração Inibidora 50 , Cinurenina/imunologia , Cinurenina/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Neoplasias/imunologia , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/química , Linfócitos T/imunologia , Triptofano/imunologia , Triptofano/metabolismo , Triptofano Oxigenase/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC50) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection.
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Understanding the intrinsic mediators that render CD8+ T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8+ T cells. Chop expression is increased in tumor-infiltrating CD8+ T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8+ T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8+ T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8+ T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Epitelial do Ovário/genética , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias Ovarianas/genética , Proteínas com Domínio T/genética , Fator de Transcrição CHOP/genética , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/patologia , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade Celular , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Knockout , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Proteínas com Domínio T/imunologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
BACKGROUND: Cancer cells are known to develop mechanisms to circumvent effective anti-tumor immunity. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to adenosine. CD39 is expressed by different immune cell populations as well as cancer cells of different tumor types and supports the tumor in escaping immune recognition and destruction. Thus, increasing extracellular ATP and simultaneously reducing adenosine concentrations in the tumor can lead to effective anti-tumor immunity. METHODS: We designed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) with specificity for human or mouse CD39 that do not need a transfection reagent or delivery system for efficient target knockdown. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and in primary human T cells. The effect of CD39 knockdown on ATP-degrading activity was evaluated by measuring levels of ATP in tumor cell supernatants and analysis of T cell proliferation in the presence of extracellular ATP. The in vivo effects of CD39-specific ASOs on target expression, anti-tumor immune responses and on tumor growth were analyzed in syngeneic mouse tumor models using multi-color flow cytometry. RESULTS: CD39-specific ASOs suppressed expression of CD39 mRNA and protein in different murine and human cancer cell lines and in primary human T cells. Degradation of extracellular ATP was strongly reduced by CD39-specific ASOs. Strikingly, CD39 knockdown by ASOs was associated with improved CD8+ T cell proliferation. Treatment of tumor-bearing mice with CD39-specific ASOs led to dose-dependent reduction of CD39-protein expression in regulatory T cells (Tregs) and tumor-associated macrophages. Moreover, frequency of intratumoral Tregs was substantially reduced in CD39 ASO-treated mice. As a consequence, the ratio of CD8+ T cells to Tregs in tumors was improved, while PD-1 expression was induced in CD39 ASO-treated intratumoral CD8+ T cells. Consequently, CD39 ASO treatment demonstrated potent reduction in tumor growth in combination with anti-PD-1 treatment. CONCLUSION: Targeting of CD39 by ASOs represents a promising state-of-the art therapeutic approach to improve immune responses against tumors.
Assuntos
Apirase/genética , Inativação Gênica , Imunidade/genética , Neoplasias/genética , Neoplasias/imunologia , Oligonucleotídeos Antissenso/genética , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Modelos Animais de Doenças , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/patologia , Oligonucleotídeos Antissenso/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-ß isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-ß immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.
Assuntos
Carcinoma/imunologia , Glioma/imunologia , Neoplasias/imunologia , Proteínas Smad/metabolismo , Fatores de Crescimento Transformadores/metabolismo , Especificidade de Anticorpos , Western Blotting , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Carcinoma/patologia , Ensaios Clínicos como Assunto , Feminino , Glioma/patologia , Humanos , Imuno-Histoquímica , Masculino , Neoplasias/metabolismo , Neoplasias/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
Transforming growth factor beta isoforms (TGF-ß1, -ß2, and -ß3) are cytokines associated with a wide range of biological processes in oncology including tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-ß signaling pathways may have a multifold therapeutic benefit for the treatment of solid tumors. Here, we describe the identification and selection processes for the development of highly potent and selective chemically modified antisense oligodeoxynucleotides (fully phosphorothioate locked nucleic acid gapmers) allowing effective and selective suppression of TGF-ß isoform expression in cell-based assays and in vivo preclinical models.
Assuntos
Terapia Genética/métodos , Neoplasias/genética , Neoplasias/terapia , Oligonucleotídeos Antissenso/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/uso terapêutico , Alanina Transaminase/sangue , Animais , Sequência de Bases , Linhagem Celular Tumoral , DNA/metabolismo , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Oligonucleotídeos Antissenso/toxicidade , Isoformas de Proteínas/genética , Isoformas de Proteínas/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Despite remarkable advances in cancer research, patients with malignant tumors such as high-grade glioma or advanced pancreatic carcinoma still face a poor prognosis. Because of the severe morbidity and mortality of such malignant tumor types, the identification of suitable molecular drug targets for causal treatment approaches is an important area of current research. Transforming growth factor-beta 2 (TGF-ß2) is an attractive target because it regulates key mechanisms of carcinogenesis, in particular immunosuppression and metastasis, and is frequently overexpressed in malignant tumors. Here we describe the development of the antisense phosphorothioate oligodeoxynucleotide trabedersen (AP 12009) which was designed for the specific inhibition of TGF-ß2 biosynthesis. In vitro and in vivo experiments confirmed the mode of action, efficacy and tolerability of trabedersen and paved the way for clinical studies. In patients with high-grade glioma, intratumoral treatment with trabedersen is currently evaluated in a pivotal, randomized and active-controlled phase III study. Intravenous application of trabedersen for the treatment of patients with advanced pancreatic carcinoma, metastasizing melanoma, or metastatic colorectal carcinoma is assessed in a currently ongoing phase I/II dose escalation study.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Tionucleotídeos/uso terapêutico , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Animais , HumanosRESUMO
Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-ß2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-ß2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF-ß2 secretion in human pancreatic cell lines with an IC50 in the low µM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF-ß2-mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell-mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen.