ABSTRACT
BACKGROUND: Hereditary angioedema is characterized by recurrent and unpredictable swellings that are disabling and potentially fatal. Selective inhibition of plasma prekallikrein production by antisense oligonucleotide treatment (donidalorsen) may reduce the frequency of attacks and the burden of disease. METHODS: In this phase 2 trial, we randomly assigned, in a 2:1 ratio, patients with hereditary angioedema with C1 inhibitor deficiency to receive four subcutaneous doses of either donidalorsen (80 mg) or placebo, with one dose administered every 4 weeks. The primary end point was the time-normalized number of investigator-confirmed angioedema attacks per month (attack rate) between week 1 (baseline) and week 17. Secondary end points included quality of life, as measured with the Angioedema Quality of Life Questionnaire (scores range from 0 to 100, with higher scores indicating worse quality of life), and safety. RESULTS: A total of 20 patients were enrolled, of whom 14 were randomly assigned to receive donidalorsen and 6 to receive placebo. The mean monthly rate of investigator-confirmed angioedema attacks was 0.23 (95% confidence interval [CI], 0.08 to 0.39) among patients receiving donidalorsen and 2.21 (95% CI, 0.58 to 3.85) among patients receiving placebo (mean difference, -90%; 95% CI, -96 to -76; P<0.001). The mean change from baseline to week 17 in the Angioedema Quality of Life Questionnaire score was -26.8 points in the donidalorsen group and -6.2 points in the placebo group (mean difference, -20.7 points; 95% CI, -32.7 to -8.7). The incidence of mild-to-moderate adverse events was 71% among patients receiving donidalorsen and 83% among those receiving placebo. CONCLUSIONS: Among patients with hereditary angioedema, donidalorsen treatment resulted in a significantly lower rate of angioedema attacks than placebo in this small, phase 2 trial. (Funded by Ionis Pharmaceuticals; ISIS 721744-CS2 ClinicalTrials.gov number, NCT04030598.).
Subject(s)
Angioedemas, Hereditary , Oligonucleotides, Antisense , Prekallikrein , Adult , Female , Humans , Male , Angioedemas, Hereditary/drug therapy , Disease-Free Survival , Drug Administration Schedule , Oligonucleotides, Antisense/adverse effects , Oligonucleotides, Antisense/therapeutic use , Patient Acuity , Prekallikrein/antagonists & inhibitors , Prekallikrein/genetics , Quality of Life , RNA, Messenger/antagonists & inhibitorsABSTRACT
Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression.
Subject(s)
Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/physiopathology , Gene Expression Regulation, Neoplastic/genetics , RNA, Long Noncoding/genetics , Animals , Cell Adhesion/genetics , Cell Movement/genetics , Disease Models, Animal , Gene Knockdown Techniques , Humans , Mice , Morphogenesis/genetics , Neoplasm Metastasis/genetics , Protein Splicing/genetics , RNA, Long Noncoding/metabolism , Signal Transduction/geneticsABSTRACT
Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.
Subject(s)
Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Mutation/drug effects , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Humans , Mice, SCID , Molecular Targeted Therapy , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Oligonucleotides, Antisense/therapeutic use , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic useABSTRACT
Pancreatic intraepithelial neoplasia is a pre-malignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2-4). So far, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavour. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression both in genetic models and in patient-derived xenografts. Specifically, we developed Msi reporter mice that allowed image-based tracking of stem cell signals within cancers, revealing that Msi expression rises as pancreatic intraepithelial neoplasia progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbour the capacity to propagate adenocarcinoma, are enriched in circulating tumour cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in the progression of pancreatic intraepithelial neoplasia to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumours, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumour penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signalling as a central regulator of pancreatic cancer.
Subject(s)
Carcinoma, Pancreatic Ductal/drug therapy , Drug Resistance, Neoplasm/drug effects , Molecular Imaging , Nerve Tissue Proteins/genetics , Pancreatic Neoplasms/drug therapy , RNA-Binding Proteins/genetics , Animals , Carcinoma in Situ/genetics , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Transformation, Neoplastic/genetics , Disease Models, Animal , Disease Progression , Drug Resistance, Neoplasm/genetics , Female , Gene Deletion , Genes, Reporter/genetics , Humans , Male , Mice , Models, Genetic , Neoplastic Cells, Circulating/metabolism , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/metabolism , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacokinetics , Oligonucleotides, Antisense/therapeutic use , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA-Binding Proteins/metabolism , Signal Transduction/drug effects , Survival Rate , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND & AIMS: Telomerase activation is the earliest event in hepatocellular carcinoma (HCC) development. Thus, we aimed to elucidate the role of telomere length maintenance during liver carcinogenesis. METHODS: Telomere length was measured in the tumor and non-tumor liver tissues of 1,502 patients (978 with HCC) and integrated with TERT alterations and expression, as well as clinical and molecular (analyzed by genome, exome, targeted and/or RNA-sequencing) features of HCC. The preclinical efficacy of anti-TERT antisense oligonucleotides (ASO) was assessed in vitro in 26 cell lines and in vivo in a xenograft mouse model. RESULTS: Aging, liver fibrosis, male sex and excessive alcohol consumption were independent determinants of liver telomere attrition. HCC that developed in livers with long telomeres frequently had wild-type TERT with progenitor features and BAP1 mutations. In contrast, HCC that developed on livers with short telomeres were enriched in the non-proliferative HCC class and frequently had somatic TERT promoter mutations. In HCCs, telomere length is stabilized in a narrow biological range around 5.7 kb, similar to non-tumor livers, by various mechanisms that activate TERT expression. Long telomeres are characteristic of very aggressive HCCs, associated with the G3 transcriptomic subclass, TP53 alterations and poor prognosis. In HCC cell lines, TERT silencing with ASO was efficient in highly proliferative and poorly differentiated cells. Treatment for 3 to 16 weeks induced cell proliferation arrest in 12 cell lines through telomere shortening, DNA damage and activation of apoptosis. The therapeutic effect was also obtained in a xenograft mouse model. CONCLUSIONS: Telomere maintenance in HCC carcinogenesis is diverse, and is associated with tumor progression and aggressiveness. The efficacy of anti-TERT ASO treatment in cell lines revealed the oncogenic addiction to TERT in HCC, providing a preclinical rationale for anti-TERT ASO treatment in HCC clinical trials. LAY SUMMARY: Telomeres are repeated DNA sequences that protect chromosomes and naturally shorten in most adult cells because of the inactivation of the TERT gene, coding for the telomerase enzyme. Here we show that telomere attrition in the liver, modulated by aging, sex, fibrosis and alcohol, associates with specific clinical and molecular features of hepatocellular carcinoma, the most frequent primary liver cancer. We also show that liver cancer is dependent on TERT reactivation and telomere maintenance, which could be targeted through a novel therapeutic approach called antisense oligonucleotides.
Subject(s)
Aging/physiology , Carcinogenesis/genetics , Carcinoma, Hepatocellular , Liver Neoplasms , Oligonucleotides, Antisense/pharmacology , Telomerase/metabolism , Telomere Homeostasis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Cell Line, Tumor , Drug Discovery , Ethanol/metabolism , Humans , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Oncogene Addiction , Sex Factors , Telomerase/genetics , Telomere Homeostasis/drug effects , Telomere Homeostasis/physiologyABSTRACT
Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.
Subject(s)
Neoplasms/genetics , Oligonucleotides, Antisense/genetics , Phosphorothioate Oligonucleotides/genetics , Proto-Oncogene Proteins p21(ras)/genetics , rab5 GTP-Binding Proteins/genetics , Endosomes/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , HT29 Cells , Humans , Neoplasms/pathology , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , Phosphorothioate Oligonucleotides/chemistry , Phosphorothioate Oligonucleotides/pharmacology , RNA, Messenger/genetics , RNA, Small Interfering/geneticsABSTRACT
Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.
Subject(s)
Autophagy/physiology , Oligonucleotides, Antisense/metabolism , Animals , Autophagosomes/metabolism , Biological Transport/physiology , Cells, Cultured , Endocytosis/physiology , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Oligonucleotides, Antisense/genetics , RNA Interference , Signal TransductionABSTRACT
Two of the most predominant features of the Alzheimer's disease (AD) brain are deposition of ß-amyloid (Aß) plaques and inflammation. The mechanism behind these pathologies remains unknown, but there is evidence to suggest that inflammation may predate the deposition of Aß. Furthermore, immune activation is increasingly being recognized as a major contributor to the pathogenesis of the disease, and disorders involving systemic inflammation, such as infection, aging, obesity, atherosclerosis, diabetes, and depression are risk factors for the development of AD. Plasminogen (PLG) is primarily a blood protein synthesized in the liver, which when cleaved into its active form, plasmin (PL), plays roles in fibrinolysis, wound healing, cell signaling, and inflammatory regulation. Here we show that PL in the blood is a regulator of brain inflammatory action and AD pathology. Depletion of PLG in the plasma of an AD mouse model through antisense oligonucleotide technology dramatically improved AD pathology and decreased glial cell activation in the brain, whereas an increase in PL activity through α-2-antiplasmin (A2AP) antisense oligonucleotide treatment exacerbated the brain's immune response and plaque deposition. These studies suggest a crucial role for peripheral PL in mediating neuroimmune cell activation and AD progression and could provide a link to systemic inflammatory risk factors that are known to be associated with AD development.
Subject(s)
Alzheimer Disease/blood , Brain/metabolism , Plasminogen/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Animals , Brain/pathology , Disease Models, Animal , Inflammation/drug therapy , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Mice , Mice, Transgenic , Oligodeoxyribonucleotides, Antisense/pharmacology , Plasminogen/antagonists & inhibitors , Plasminogen/geneticsABSTRACT
BACKGROUND: Adult T-cell leukemia lymphoma (ATLL) is a chemotherapy-resistant malignancy with a median survival of less than one year that will afflict between one hundred thousand and one million individuals worldwide who are currently infected with human T-cell leukemia virus type 1. Recurrent somatic mutations in host genes have exposed the T-cell receptor pathway through nuclear factor κB to interferon regulatory factor 4 (IRF4) as an essential driver for this malignancy. We sought to determine if IRF4 represents a therapeutic target for ATLL and to identify downstream effectors and biomarkers of IRF4 signaling in vivo. RESULTS: ATLL cell lines, particularly Tax viral oncoprotein-negative cell lines, that most closely resemble ATLL in humans, were sensitive to dose- and time-dependent inhibition by a next-generation class of IRF4 antisense oligonucleotides (ASOs) that employ constrained ethyl residues that mediate RNase H-dependent RNA degradation. ATLL cell lines were also sensitive to lenalidomide, which repressed IRF4 expression. Both ASOs and lenalidomide inhibited ATLL proliferation in vitro and in vivo. To identify biomarkers of IRF4-mediated CD4 + T-cell expansion in vivo, transcriptomic analysis identified several genes that encode key regulators of ATLL, including interleukin 2 receptor subunits α and ß, KIT ligand, cytotoxic T-lymphocyte-associated protein 4, and thymocyte selection-associated high mobility group protein TOX 2. CONCLUSIONS: These data support the pursuit of IRF4 as a therapeutic target in ATLL with the use of either ASOs or lenalidomide.
Subject(s)
HTLV-I Infections/metabolism , Interferon Regulatory Factors/metabolism , Leukemia-Lymphoma, Adult T-Cell/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Products, tax/metabolism , HTLV-I Infections/drug therapy , HTLV-I Infections/pathology , Human T-lymphotropic virus 1 , Humans , Interferon Regulatory Factors/genetics , Lenalidomide/pharmacology , Leukemia-Lymphoma, Adult T-Cell/drug therapy , Leukemia-Lymphoma, Adult T-Cell/pathology , Mice , Oligonucleotides, Antisense/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Thionucleotides/pharmacologyABSTRACT
Sepsis causes an activation of the human contact system, an inflammatory response mechanism against foreign surfaces, proteins and pathogens. The serine proteases of the contact system, factor XII and plasma kallikrein, are decreased in plasma of septic patients, which was previously associated with an unfavorable outcome. However, the precise mechanisms and roles of contact system factors in bacterial sepsis are poorly understood. We, therefore, studied the physiological relevance of factor XII and plasma kallikrein in a mouse model of experimental sepsis. We show that decreased plasma kallikrein concentration in septic mice is a result of reduced mRNA expression plasma prekallikrein gene, indicating that plasma kallikrein belong to negative acute phase proteins. Investigations regarding the pathophysiological function of contact system proteases during sepsis revealed different roles for factor XII and plasma kallikrein. In vitro, factor XII decelerated bacteria induced fibrinolysis, whereas plasma kallikrein supported it. Remarkably, depletion of plasma kallikrein (but not factor XII) by treatment with antisense-oligonucleotides, dampens bacterial dissemination and growth in multiple organs in the mouse sepsis model. These findings identify plasma kallikrein as a novel host pathogenicity factor in Streptococcus pyogenes sepsis.
Subject(s)
Sepsis , Streptococcal Infections , Animals , Factor XII , Humans , Mice , Peptide HydrolasesABSTRACT
Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.
Subject(s)
Acetylgalactosamine/chemistry , Gene Transfer Techniques , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/chemistry , Animals , Asialoglycoprotein Receptor/genetics , Asialoglycoprotein Receptor/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line , Cells, Cultured , Gene Expression , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , MiceABSTRACT
BACKGROUND: PVT1 has emerged as an oncogene in many tumor types. However, its role in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) is unknown. The aim of this study was to assess the role of PVT1 in BE/EAC progression and uncover its therapeutic value against EAC. METHODS: PVT1 expression was assessed by qPCR in normal, BE, and EAC tissues and statistical analysis was performed to determine the association of PVT1 expression and EAC (stage, metastases, and survival). PVT1 antisense oligonucleotides (ASOs) were tested for their antitumor activity in vitro and in vivo. RESULTS: PVT1 expression was up-regulated in EACs compared with paired BEs, and normal esophageal tissues. High expression of PVT1 was associated with poor differentiation, lymph node metastases, and shorter survival. Effective knockdown of PVT1 in EAC cells using PVT1 ASOs resulted in decreased cell proliferation, invasion, colony formation, tumor sphere formation, and reduced proportion of ALDH1A1+ cells. Mechanistically, we discovered mutual regulation of PVT1 and YAP1 in EAC cells. Inhibition of PVT1 by PVT1 ASOs suppressed YAP1 expression through increased phosphor-LATS1and phosphor-YAP1 while knockout of YAP1 in EAC cells significantly suppressed PVT1 levels indicating a positive regulation of PVT1 by YAP1. Most importantly, we found that targeting both PVT1 and YAP1 using their specific ASOs led to better antitumor activity in vitro and in vivo. CONCLUSIONS: Our results provide strong evidence that PVT1 confers an aggressive phenotype to EAC and is a poor prognosticator. Combined targeting of PVT1 and YAP1 provided the highest therapeutic index and represents a novel therapeutic strategy.
Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/mortality , Biomarkers, Tumor , Esophageal Neoplasms/genetics , Esophageal Neoplasms/mortality , Gene Expression Regulation, Neoplastic , RNA, Long Noncoding/genetics , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adenocarcinoma/drug therapy , Animals , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Disease Models, Animal , Esophageal Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Models, Biological , Prognosis , Transcription Factors/antagonists & inhibitors , YAP-Signaling ProteinsABSTRACT
Vascular abnormalities and inflammation are found in many Alzheimer disease (AD) patients, but whether these changes play a causative role in AD is not clear. The factor XII (FXII) -initiated contact system can trigger both vascular pathology and inflammation and is activated in AD patients and AD mice. We have investigated the role of the contact system in AD pathogenesis. Cleavage of high-molecular-weight kininogen (HK), a marker for activation of the inflammatory arm of the contact system, is increased in a mouse model of AD, and this cleavage is temporally correlated with the onset of brain inflammation. Depletion of FXII in AD mice inhibited HK cleavage in plasma and reduced neuroinflammation, fibrinogen deposition, and neurodegeneration in the brain. Moreover, FXII-depleted AD mice showed better cognitive function than untreated AD mice. These results indicate that FXII-mediated contact system activation contributes to AD pathogenesis, and therefore this system may offer novel targets for AD treatment.
Subject(s)
Alzheimer Disease , Brain , Cognitive Dysfunction , Factor XII/metabolism , Vascular Diseases , Alzheimer Disease/blood , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Alzheimer Disease/physiopathology , Animals , Brain/blood supply , Brain/metabolism , Brain/pathology , Brain/physiopathology , Cognitive Dysfunction/blood , Cognitive Dysfunction/genetics , Cognitive Dysfunction/pathology , Cognitive Dysfunction/physiopathology , Kininogen, High-Molecular-Weight/blood , Mice , Mice, Transgenic , Vascular Diseases/blood , Vascular Diseases/genetics , Vascular Diseases/pathology , Vascular Diseases/physiopathologyABSTRACT
Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUG(exp)) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG(exp) RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.
Subject(s)
Cell Nucleus/genetics , Gene Silencing , Myotonic Dystrophy/genetics , Myotonic Dystrophy/therapy , RNA/antagonists & inhibitors , RNA/genetics , Alleles , Animals , Base Sequence , Cell Nucleus/drug effects , Disease Models, Animal , Gene Knockdown Techniques , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Transgenic , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Myotonic Dystrophy/pathology , Myotonic Dystrophy/physiopathology , Myotonin-Protein Kinase , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , Protein Serine-Threonine Kinases/genetics , RNA/metabolism , RNA, Long Noncoding , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Untranslated/genetics , Ribonuclease H/metabolism , Transcriptome/drug effects , Transcriptome/genetics , Trinucleotide Repeat Expansion/geneticsABSTRACT
Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 is caused by an expanded CTG repeat in the 3'-untranslated region of DMPK, the gene encoding dystrophia myotonica protein kinase (DMPK). Antisense oligonucleotides (ASOs) containing 2',4'-constrained ethyl-modified (cEt) residues exhibit a significantly increased RNA binding affinity and in vivo potency relative to those modified with other 2'-chemistries, which we speculated could translate to enhanced activity in extrahepatic tissues, such as muscle. Here, we describe the design and characterization of a cEt gapmer DMPK ASO (ISIS 486178), with potent activity in vitro and in vivo against mouse, monkey, and human DMPK. Systemic delivery of unformulated ISIS 486718 to wild-type mice decreased DMPK mRNA levels by up to 90% in liver and skeletal muscle. Similarly, treatment of either human DMPK transgenic mice or cynomolgus monkeys with ISIS 486178 led to up to 70% inhibition of DMPK in multiple skeletal muscles and â¼50% in cardiac muscle in both species. Importantly, inhibition of DMPK was well tolerated and was not associated with any skeletal muscle or cardiac toxicity. Also interesting was the demonstration that the inhibition of DMPK mRNA levels in muscle was maintained for up to 16 and 13 weeks post-treatment in mice and monkeys, respectively. These results demonstrate that cEt-modified ASOs show potent activity in skeletal muscle, and that this attractive therapeutic approach warrants further clinical investigation to inhibit the gain-of-function toxic RNA underlying the pathogenesis of DM1.
Subject(s)
Myotonic Dystrophy/drug therapy , Myotonin-Protein Kinase/metabolism , Oligonucleotides, Antisense/pharmacology , Oligonucleotides/pharmacology , Animals , Cell Line , Humans , Macaca fascicularis , Male , Mice , Mice, Transgenic , Muscle, Skeletal/enzymology , Myotonin-Protein Kinase/antagonists & inhibitors , Myotonin-Protein Kinase/genetics , Oligonucleotides/chemistry , RNA, Messenger/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.
Subject(s)
Blood Coagulation/drug effects , Factor XI/metabolism , Fibrinolytic Agents/administration & dosage , Oligonucleotides, Antisense/administration & dosage , Thrombosis/prevention & control , Animals , Antibodies, Monoclonal/administration & dosage , Arteriovenous Shunt, Surgical , Bleeding Time , Collagen , Disease Models, Animal , Dose-Response Relationship, Drug , Down-Regulation , Factor XI/antagonists & inhibitors , Factor XI/genetics , Fibrinolytic Agents/toxicity , Hemorrhage/chemically induced , Macaca fascicularis , Oligonucleotides, Antisense/toxicity , Papio , Thrombin/metabolism , Thrombosis/blood , Thrombosis/etiology , Thrombosis/genetics , Time FactorsABSTRACT
Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had â¼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.
Subject(s)
Factor XII Deficiency/blood , Hemorrhage/blood , Hemorrhage/etiology , Prekallikrein/deficiency , Thrombosis/blood , Thrombosis/prevention & control , Animals , Disease Models, Animal , Factor XII/antagonists & inhibitors , Factor XII/genetics , Factor XII Deficiency/genetics , Gene Knockdown Techniques , Hemorrhage/genetics , Hemostasis/genetics , Hemostasis/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/genetics , Prekallikrein/antagonists & inhibitors , Prekallikrein/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Risk Factors , Thrombosis/geneticsABSTRACT
Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A. In contrast, targeted disruption of DNMT1 alleles in HCT116 human colon cancer cells produced clones that retained CpG island methylation and associated tumor-suppressor gene silencing, whereas HCT116 clones with inactivation of both DNMT1 and DNMT3B showed much lower levels of DNA methylation, suggesting that the two enzymes are highly cooperative. We used a combination of genetic (antisense and siRNA) and pharmacologic (5-aza-2'-deoxycytidine) inhibitors of DNA methyl transferases to study the contribution of the DNMT isotypes to cancer-cell methylation. Selective depletion of DNMT1 using either antisense or siRNA resulted in lower cellular maintenance methyltransferase activity, global and gene-specific demethylation and re-expression of tumor-suppressor genes in human cancer cells. Specific depletion of DNMT1 but not DNMT3A or DNMT3B markedly potentiated the ability of 5-aza-2'-deoxycytidine to reactivate silenced tumor-suppressor genes, indicating that inhibition of DNMT1 function is the principal means by which 5-aza-2'-deoxycytidine reactivates genes. These results indicate that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells.