Antisense Oligonucleotide-based Therapeutics Research Articles

Using RNA-seq to Assess Off-Target Effects of Antisense Oligonucleotides in Human Cell Lines.

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. 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. 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. 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. 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.

Angubindin-1 opens the blood–brain barrier in vivo for delivery of antisense oligonucleotide to the central nervous system

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood–brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood–brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood–brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood–brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders.

ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3′-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3′-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.

New Approach for Antisense Oligonucleotide-Mediated Exon Skipping in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a lethalmuscle disorder characterized by mutations in the DMD gene. These mutations primarily disrupt the reading frame, resulting in the absence of functional dystrophin protein. Exon skipping, which involves the use of antisense oligonucleotides is a promising therapeutic approach for DMD, and clinical trials on exon skipping are currently underway in DMD patients. Recently, stable and less-toxic antisense oligonucleotides with higher efficacy have been developed in mouse and dog models of DMD. This review highlights a new approach for antisense oligonucleotide-based therapeutics for DMD, particularly for exon skipping-based methods.

Special Issue on Modeling and Simulation

Special Issue on Modeling and Simulation

Antisense oligonucleotide-based therapeutics for cancer.

There has been steady progress in antisense technology over the past 14 years. We now have a far better appreciation of the attributes and limitations of the technology. Antisense oligonucleotides have been used to selectively inhibit thousands of genes in mammalian cells, hundreds, if not thousands, of genes in rodents and other species and multiple genes in humans. There are over 20 antisense drugs currently in clinical trials, several of which are showing promising results. Like any other class of drugs in development, there will continue to be successes and failures in the clinic. Despite some disappointments with the technology, it appears to be a valid platform for both drug discovery and as an experimental tool for functionalizing genes. Advances in the medicinal chemistry and formulation of antisense oligonucleotides will further enhance their therapeutic and commercial potential.

MONITORING GENE EXPRESSION IN LIVING ANIMALS USING BIOLUMINESCENT REPORTERS.• 844

The introduction of nucleic acid sequences into the cells of living animals is the basis of generating transgenic and chimeric animals, which themselves serve as basic tools in genetics and as a foundation for emerging gene therapies, gene vaccines, and antisense oligonucleotide-based therapeutics. The detection of gene-modifying events now requires that large numbers of cells be altered, selected, grown, and analyzed in a time-consuming and labor-intensive sequence of steps. A method for rapid, in situ assessment of the uptake and expression of nucleic acids would thus facilitate evaluation of gene delivery systems and DNA-based therapies. We report on development of a real-time bioluminescent reporter that noninvasively indicates the level of promoter activity in living animals. Such a system differs from existing green-fluorescent-protein-(GFP)-based systems, as our reporters spontaneously emit light without a need for outside light sources. A gene fusion product consisting of HIV-1 LTR (promoter) and firefly luciferase gene complex (reporter) was studied in a transgenic mouse. Photons from thein vivo luciferase reaction were detected by a CCD camera, after transmission through the animal's tissues, and used as an indication of the level of gene expression. This allowed us to estimate, in real time, the extent of promoter activity. We were able to assess activity, both on the surface of the animal as well as in deep tissues. In the skin of living transgenic mice, we assessed the levels of induction of HIV-LTR in response to various stimuli (in murine and human tissues, the HIV-1 LTR promoter can be activated by chemical, photo, and thermal signals). Reduction of photon emission from luciferase expressed in the skin was observed during ATP depletion, suggesting use of bioluminescence as an in situ ATP sensor. Photons emitted from internal organs were also detected externally, indicating that a wide variety of promoters expressed at various tissues sites may be studied using this approach. Use of such a reporter system may facilitate an in vivo assessment of new therapies targeting regulation of viral and host gene expression. Our approach has advantages over dye-producing methods (e.g., GFP), as a low background signal makes near single-event detection possible, the signal is real-time rather than integrated, and no cytotoxic photosensitizing dyes are produced. These studies open a window through which biological processes can be viewed in vivo, illuminating the temporal and spatial distribution of gene expression in animals and humans. Supported by NIH N43-NS-4-2315 & RR-00081, Universitywide AIDS Prog., Packard Foundation (Stanford), and ONR N-00014-94-1-1024.

Recent developments in peptide drug delivery to the brain.

Peptide-based therapeutics are highly water-soluble compounds that do not readily enter brain from blood owing to poor transport through the brain capillary endothelial wall, i.e., the blood-brain barrier (BBB). Strategies available for peptide drug delivery to brain include: (a) neurosurgical-based (intraventricular drug infusion, hyperosmotic opening of the BBB); (b) pharmacological-based (peptide lipidization, liposomes); and (c) physiological-based (biochemical opening of the BBB, chimeric peptides). Chimeric peptides are formed by the covalent coupling of a pharmaceutical peptide (that is normally not transported through the BBB) to a brain transport vector that undergoes absorptive-mediated or receptor-mediated transcytosis through the BBB. The most efficient brain transport vector known to date is a monoclonal antibody to the transferrin receptor, and this vector achieves a brain volume of distribution approximately 18-fold greater than the plasma space by 5 hr after a single intravenous injection of antibody. The chimeric peptides are formed generally with chemical-based linkers. However, avidin/biotin-based linkers allow for high yield coupling of drug to vector, and for the release of biologically-active peptide following cleavage of the chimeric peptide linker. These strategies may also be used for the delivery of antisense oligonucleotide-based therapeutics to brain. In conclusion, the development of efficacious neuropharmaceuticals in the future will require the development of both drug delivery and drug discovery strategies that operate in parallel.