Antisense oligonucleotide-mediated inhibition of angiopoietin-like protein 3 increases reverse cholesterol transport in mice.

Supported by an abundance of experimental and genetic evidence, angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for cardiovascular disease. ANGPTL3 is primarily produced by the liver and is a potent modulator of plasma lipids and lipoproteins. Experimental models and subjects with loss-of-function Angptl3 mutations typically present with lower levels of HDL-C than noncarriers.

Characterization of the Activity and Distribution of a 2'-O-Methoxyethyl-Modified Antisense Oligonucleotide in Models of Acute and Chronic Kidney Disease.

To determine if the pharmacokinetics and pharmacodynamics of gapmer antisense oligonucleotides (ASOs), containing phosphorothioate backbones and 2'-O-methoxyethyl RNA modifications (2'-MOE ASOs), can be altered by renal disease, a series of experiments were performed in models of chronic kidney disease (CKD) and acute kidney injury (AKI). In an adenine diet model of CKD, 2'-MOE ASO activity in the whole kidney was preserved and the reduction in target RNA was sustained for 2-4 weeks postdose. Additionally, 2'-MOE ASO distribution within the kidney was altered in mice with CKD, in that ASO delivery to cortical regions with tubular damage was reduced while distribution to the medulla was increased.

Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver.

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur.

Co-Administration of an Excipient Oligonucleotide Helps Delineate Pathways of Productive and Nonproductive Uptake of Phosphorothioate Antisense Oligonucleotides in the Liver.

Phosphorothioate (PS) modified antisense oligonucleotides (ASOs) have progressed rapidly in the clinic for treating a variety of disease indications. We previously demonstrated that the activity of PS ASOs in the liver can be enhanced by co-infusion of an excipient oligonucleotide (EON). It was posited that the EON saturates a nonproductive uptake pathway(s) thereby permitting accumulation of the PS ASO in a productive tissue compartment.

Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor.

Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied.

Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver.

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs.

CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation.

Preclinical and clinical data suggest CD40 activation contributes to renal inflammation and injury. We sought to test whether upregulation of CD40 in the kidney is a causative factor of renal pathology and if reduction of renal CD40 expression, using antisense oligonucleotides (ASOs) targeting CD40, would be beneficial in mouse models of glomerular injury and unilateral ureter obstruction. Administration of a Generation 2.

CDK8: a positive regulator of transcription.

CDK8 belongs to a group of cyclin-dependent kinases involved in transcriptional regulation from yeast to mammals. CDK8 associates with the Mediator complex, but functions outside of Mediator are also likely. Historically, CDK8 has been described mostly as a transcriptional repressor, but a growing body of research provides unequivocal evidence for various roles of CDK8 in gene activation.

CDK8 is a positive regulator of transcriptional elongation within the serum response network.

The Mediator complex allows communication between transcription factors and RNA polymerase II (RNAPII). Cyclin-dependent kinase 8 (CDK8), the kinase found in some variants of Mediator, has been characterized mostly as a transcriptional repressor. Recently, CDK8 was demonstrated to be a potent oncoprotein.

A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint.

We reported previously that when cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. Here we show that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3' end of the p21 gene.

The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator.

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase.

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3.

The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex.

Stimulus-specific transcriptional regulation within the p53 network.

The p53 transcriptional network is composed of hundreds of effector genes involved in varied stress-response pathways, including cell cycle arrest and apoptosis. It is not clear how distinct p53 target genes are differentially activated to trigger stress-specific biological responses. We analyzed the p53 transcriptional program upon activation by two DNA-damaging agents, UVC and doxorubicin, versus the non-genotoxic molecule Nutlin-3.

CDK8 is a stimulus-specific positive coregulator of p53 target genes.

The p53 transcriptional network orchestrates alternative stress responses such as cell-cycle arrest and apoptosis. Here we investigate the mechanism of differential expression of p21, a key mediator of p53-dependent cell-cycle arrest. We demonstrate that the transcriptional activity of the p21 promoter varies greatly in response to distinct p53-activating stimuli.

Activated protein C signals through the thrombin receptor PAR1 in endothelial cells.

The anti-inflammatory effects of activated protein C (APC) have lead to its recent approval for the treatment of sepsis. Although the endothelial cell protein C receptor (EPCR) plays a crucial role in APC's protective roles in septicemia, the precise signaling mechanism of the protease APC remains unclear. In fibroblast overexpression systems, we find that APC activates protease activated receptors (PAR) 1 and 2 in an EPCR-dependent manner.

Activation of endothelial cell protease activated receptor 1 by the protein C pathway.

The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2.