Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells.

BACKGROUND: Colon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated. METHODS: In present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot. RESULTS: We found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered. CONCLUSIONS: Therefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.

RNA therapeutics for ß-thalassemia.

ß-thalassemia is an autosomal recessive disease, caused by one or more mutations in the ß-globin gene that reduces or abolishes ß-globin chain synthesis causing an imbalance in the ratio of α- and ß-globin chain. Therefore, the ability to target mutations will provide a good result in the treatment of ß-thalassemia. RNA therapeutics represents a promising class of drugs inclusive antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA) and APTAMER have investigated in clinical trials for treatment of human diseases as ß-thalassemia; Especially, ASO therapeutics can completely treat ß-thalassemia patients by the way of making ASO infiltrating through erythrocyte progenitor cells, migrating to the nucleus and hybridizing with abnormal splicing sites to suppress an abnormal splicing pattern of ß-globin pre-mRNA. As a result, the exactly splicing process is restored to increase the expression of ß-globin which increases the amount of mature hemoglobin of red blood cells of ß-thalassemia patients. Furthermore, current study demonstrates that RNA-based therapeutics get lots of good results for ß-thalassemia patients. Then, this chapter focuses on current advances of RNA-based therapeutics and addresses current challenges with their development and application for treatment of ß-thalassemia patients.

Addressing residual risk beyond statin therapy: New targets in the management of dyslipidaemias-A report from the European Society of Cardiology Cardiovascular Round Table.

Cardiovascular (CV) disease is the most common cause of death in Europe. Despite proven benefits, use of lipid-lowering therapy remains suboptimal. Treatment goals are often not achieved, even in patients at high risk with atherosclerotic CV disease (ASCVD). The occurrence of CV events in patients on lipid-lowering drugs is defined as "residual risk", and can result from inadequate control of plasma lipids or blood pressure, inflammation, diabetes, and environmental hazards. Assessment of CV risk factors and vascular imaging can aid in the evaluation and management decisions for individual patients. Lifestyle measures remain the primary intervention for lowering CV risk. Where drug therapies are required to reach lipid treatment targets, their effectiveness increases when they are combined with lifestyle measures delivered through formal programs. However, lipid drug dosage and poor adherence to treatment remain major obstacles to event-free survival. This article discusses guideline-supported treatment algorithms beyond statin therapy that can help reduce residual risk in specific patient profiles while also likely resulting in substantial healthcare savings through better patient management and treatment adherence.

Deep screening of proximal and distal splicing-regulatory elements in a native sequence context.

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is impeded by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identified not only known SREs, but also a novel distal intronic splicing enhancer, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Triazole-linked nucleic acids: Synthesis, therapeutics and synthetic biology applications.

This article covers the triazole-linked nucleic acids where the triazole linkage (TL) replaces the natural phosphate backbone. The replacement is done at either a few selected linkages or all the phosphate linkages. Two triazole linkages, the four-atom TL1 and the six-atom TL2, have been discussed in detail. These triazole-modified oligonucleotides have found a wide range of applications, from therapeutics to synthetic biology. For example, the triazole-linked oligonucleotides have been used in the antisense oligonucleotide (ASO), small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology as therapeutic agents. Due to the ease of the synthesis and a wide range of biocompatibility, the triazole linkage TL2 has been used to assemble a functional 300-mer DNA from alkyne- and azide-functionalized 100-mer oligonucleotides as well as an epigenetically modified variant of a 335 base-pair gene from ten short oligonucleotides. These outcomes highlight the potential of triazole-linked nucleic acids and open the doors for other TL designs and artificial backbones to fully exploit the vast potential of artificial nucleic acids in therapeutics, synthetic biology and biotechnology.

Making Sense of Antisense Oligonucleotide Therapeutics Targeting Bcl-2.

The B-cell lymphoma 2 (Bcl-2) family, comprised of pro- and anti-apoptotic proteins, regulates the delicate balance between programmed cell death and cell survival. The Bcl-2 family is essential in the maintenance of tissue homeostasis, but also a key culprit in tumorigenesis. Anti-apoptotic Bcl-2, the founding member of this family, was discovered due to its dysregulated expression in non-Hodgkin's lymphoma. Bcl-2 is a central protagonist in a wide range of human cancers, promoting cell survival, angiogenesis and chemotherapy resistance; this has prompted the development of Bcl-2-targeting drugs. Antisense oligonucleotides (ASO) are highly specific nucleic acid polymers used to modulate target gene expression. Over the past 25 years several Bcl-2 ASO have been developed in preclinical studies and explored in clinical trials. This review will describe the history and development of Bcl-2-targeted ASO; from initial attempts, optimizations, clinical trials undertaken and the promising candidates at hand.

Preclinical Evaluation of the Renal Toxicity of Oligonucleotide Therapeutics in Mice.

Antisense oligonucleotides (ASO) therapeutics hold great promise for the treatment of numerous diseases, and several ASO drugs have now reached market approval, confirming the potential of this approach. However, some candidates have also failed, due to limited biodistribution/uptake and poor safety profile. In pursuit of better delivery and higher cellular uptake, ASO are being optimized, and new chemistries are developed or conjugated with various ligands. While these developments may lead to candidates with higher potency, it is important to keep the safety aspects in sight and screen for potential toxicity in early phases of preclinical development to avoid subsequent failure in clinical development. Our understanding of ASO-mediated toxicity keeps improving with increased preclinical and clinical data available. In this chapter, we will focus on the assessment of renal toxicity in mice and describe methods to measure the levels of general urinary biomarkers as well as acute kidney injury biomarkers following ASO treatment.

Long Non-coding RNAs With In Vitro and In Vivo Efficacy in Preclinical Models of Esophageal Squamous Cell Carcinoma Which Act by a Non-microRNA Sponging Mechanism.

Esophageal squamous cell carcinoma is a type of cancer with dismal prognosis. Surgery, chemo- and radiation therapy, as well as immune checkpoint-blocking immunotherapy lead to limited improvement of survival of patients; therapy resistance and recurrencies hamper these treatment modalities. Therefore, the identification of new targets and treatment approaches is of paramount importance. We have searched the literature and identified 7 down-regulated and 16 up-regulated non-coding RNAs, which showed efficacy in preclinical esophageal squamous cell carcinoma-related in vitro and in vivo models, and discuss their diverse mode of actions. We excluded long non-coding RNAs, which act by sponging of microRNAs. It is presently unclear whether long non-coding RNA/protein, DNA and RNA interactions can be targeted with small molecules. We describe reconstitution therapy and inhibition of the corresponding long non-coding RNAs with small interfering RNAs and antisense oligonucleotides. Also, we discuss emerging targets for treatment of esophageal squamous cell carcinoma.

LncRNAs as Therapeutic Targets for Autophagy-involved Cardiovascular Diseases: A Review of Molecular Mechanism and T herapy Strategy.

BACKGROUND: Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. The concept of precision medicine in CVD therapy today requires the incorporation of individual genetic and environmental variability to achieve personalized disease prevention and tailored treatment. Autophagy, an evolutionarily conserved intracellular degradation process, has been demonstrated to be essential in the pathogenesis of various CVDs. Nonetheless, there have been no effective treatments for autophagy- involved CVDs. Long noncoding RNAs (lncRNAs) are noncoding RNA sequences that play versatile roles in autophagy regulation, but much needs to be explored about the relationship between lncRNAs and autophagy-involved CVDs. SUMMARY: Increasing evidence has shown that lncRNAs contribute considerably to modulate autophagy in the context of CVDs. In this review, we first summarize the current knowledge of the role lncRNAs play in cardiovascular autophagy and autophagy-involved CVDs. Then, recent developments of antisense oligonucleotides (ASOs) designed to target lncRNAs to specifically modulate autophagy in diseased hearts and vessels are discussed, focusing primarily on structure-activity relationships of distinct chemical modifications and relevant clinical trials. PERSPECTIVE: ASOs are promising in cardiovascular drug innovation. We hope that future studies of lncRNA-based therapies would overcome existing technical limitations and help people who suffer from autophagy-involved CVDs.

Overcoming the challenges of tissue delivery for oligonucleotide therapeutics.

Synthetic therapeutic oligonucleotides (STO) represent the third bonafide platform for drug discovery in the pharmaceutical industry after small molecule and protein therapeutics. So far, thirteen STOs have been approved by regulatory agencies and over one hundred of them are in different stages of clinical trials. STOs hybridize to their target RNA or DNA in cells via Watson-Crick base pairing to exert their pharmacological effects. This unique class of therapeutic agents has the potential to target genes and gene products that are considered undruggable by other therapeutic platforms. However, STOs must overcome several extracellular and intracellular obstacles to interact with their biological RNA targets inside cells. These obstacles include degradation by extracellular nucleases, scavenging by the reticuloendothelial system, filtration by the kidney, traversing the capillary endothelium to access the tissue interstitium, cell-surface receptor-mediated endocytic uptake, and escape from endolysosomal compartments to access the nuclear and/or cytoplasmic compartments where their targets reside. In this review, we present the recent advances in this field with a specific focus on antisense oligonucleotides (ASOs) and siRNA therapeutics.

Knockdown of Z Mutant Alpha-1 Antitrypsin In Vivo Using Modified DNA Antisense Oligonucleotides.

Alpha-1 antitrypsin (AAT) is a serum protease inhibitor, mainly expressed in and secreted from hepatocytes, important for regulating neutrophil elastase activity among other proteases. Various mutations in AAT cause alpha-1 antitrypsin deficiency (AATD), a rare hereditary disorder that results in liver disease due to accumulation of AAT aggregates and lung disease from excessive neutrophil elastase activity. PiZ transgenic mice contain the human AAT genomic region harboring the most common AATD mutation, the Glu342Lys (Z) point mutation. These mice effectively recapitulate the liver disease exhibited in AATD patients, including AAT protein aggregates, hepatocyte death, and eventual liver fibrosis. Previously, we demonstrated that modified antisense oligonucleotides (ASOs) can dramatically reduce Z-AAT RNA and protein levels in PiZ mice enabling inhibition, prevention, and reversal of the associated liver disease. Here, we describe in detail usage of AAT-ASOs to knock down Z-AAT in PiZ mice with a focus on preparation and in vivo delivery of ASOs, as well as detailed workflows pertaining to the analysis of Z-AAT mRNA, plasma protein, and soluble/insoluble liver protein levels following ASO administration.

First proof of pharmacology in humans of a novel glucagon receptor antisense drug.

Fasting and postprandial hyperglucagonemia in type 2 diabetes mellitus (T2DM) patients cause excessive hepatic glucose production (HGP), suggesting that attenuation of hepatic glucagon action could be a therapeutic strategy for T2DM. In this study we evaluated the safety, tolerability, PK, and pharmacodynamics in healthy human volunteers of single and multiple doses (50-400 mg) ISIS 325568, a 2'-O-MOE antisense (ASO) developed to reduce hepatic glucagon receptor (GCGR) mRNA expression. In the multiple dose cohorts, treatment consisted of eight doses of ISIS 325568 or placebo over 6-weeks. Drug effects were assessed using serial fasting glucagon measurements and the glycemic response to a glucagon challenge at baseline and at the end of 6-week treatment. ISIS 325568 was not associated with clinically relevant changes. Dose-dependent predominantly mild injection site reactions were the most common side-effect. Active treatment caused a gradual increase in fasting glucagon levels and, compared to placebo, a significantly blunted glucagon-induced increase in plasma glucose AUC (24%, P < 0.0001) and HGP (13%, P = 0.007) at the 400 mg/week dose. Six weeks treatment with ISIS 325568 in healthy volunteers attenuated glucagon-stimulated HGP and glucose excursions, supporting further evaluation of the GCGR antisense approach in patients with T2DM.