Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies.

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3' UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

Acute Neurotoxicity of Antisense Oligonucleotides After Intracerebroventricular Injection Into Mouse Brain Can Be Predicted from Sequence Features.

Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of in vitro, in vivo, and bioinformatics approaches to identify a sequence design algorithm, which can reduce the number of acutely toxic molecules synthesized and tested in mice. We find a cellular assay measuring spontaneous calcium oscillations in neuronal cells can predict the behavioral side effects after ICV dosing, and may provide a mechanistic explanation for these observations. We identify sequence features that are overrepresented or underrepresented among oligonucleotides causing these reductions in calcium oscillations. A weighted linear combination of the five most informative sequence features predicts the outcome of ICV dosing with >80% accuracy. From this, we develop a bioinformatics tool that allows oligonucleotide designs with acceptable acute neurotoxic potential to be identified, thereby reducing the number of toxic molecules entering drug discovery pipelines. The informative sequence features we identified also suggest areas in which to focus future medicinal chemistry efforts.

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers.

Phosphorothioate (PS) modification of antisense oligonucleotides (ASOs) is a critical factor enabling their therapeutic use. Standard chemical synthesis incorporates this group in a stereorandom manner; however, significant effort was made over the years to establish and characterize the impact of chiral control. In this work, we present our in-depth characterization of interactions between Escherichia coli RNase H and RNA-DNA heteroduplexes carrying chirally defined PS groups. First, using a massive parallel assay, we showed that at least a single Rp-PS group is necessary for efficient RNase H-mediated cleavage. We followed by demonstrating that this group needs to be aligned to the phosphate-binding pocket of RNase H, and that chiral status of other PS groups in close proximity to RNase H does not affect cleavage efficiency. We have shown that RNase H's PS chiral preference can be utilized to guide cleavage to a specific chemical bond. Finally, we present a strategy for ASO optimization by mapping preferred RNase H cleavage sites of a non-thioated compound, followed by introduction of Rp-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an Sp-PS at the same location.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides.

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Chemical Diversity of Locked Nucleic Acid-Modified Antisense Oligonucleotides Allows Optimization of Pharmaceutical Properties.

The identification of molecules that can modulate RNA or protein function and the subsequent chemical and structural optimization to refine such molecules into drugs is a key activity in drug discovery. Here, we explored the extent to which chemical and structural differences in antisense oligonucleotides, designed as gapmers and capable of recruiting RNase H for target RNA cleavage, can affect their functional properties. To facilitate structure-activity learning, we analyzed two sets of iso-sequential locked nucleic acid (LNA)-modified gapmers, where we systematically varied the number and positions of LNA modifications in the flanks. In total, we evaluated 768 different and architecturally diverse gapmers in HeLa cells for target knockdown activity and cytotoxic potential and found widespread differences in both of these properties. Binding affinity between gapmer and RNA target, as well as the presence of certain short sequence motifs in the gap region, can explain these differences, and we propose statistical and machine-learning models that can be used to predict region-specific, optimal LNA-modification architectures. Once accessible regions in the target of interest have been identified, our results show how to refine and optimize LNA gapmers with improved pharmacological profiles targeting such regions.

Identifying Suitable Target Regions and Analyzing Off-Target Effects of Therapeutic Oligonucleotides.

Antisense oligonucleotides (AONs) that promote degradation of complementary RNA are being developed as therapeutics. Here, we describe a simple computational workflow for identification of the regions on an RNA that are suitable for targeting with such AONs. The workflow is based on the statistical programming language R, and the calculations and data processing can be carried out on a desktop computer. Our workflow integrates well-established data resources and RNA structure-prediction tools and can be modified easily and expanded as new resources become available.

Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice.

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.

A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides.

The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (Tm) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.

Locked nucleic acid: modality, diversity, and drug discovery.

Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with improved pharmacological properties in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity and potency, is widely used for this purpose. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide is a unique entity, and small structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.

RNase H sequence preferences influence antisense oligonucleotide efficiency.

RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA-DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides. The sequences preferred by HIV-1 RNase H are distributed in the HIV genome in a way suggesting selection for efficient RNA cleavage during replication. Our findings can be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H may have shaped evolution of the viral genome and contributed to the use of tRNA-Lys3 as primer during viral replication.

Managing the sequence-specificity of antisense oligonucleotides in drug discovery.

All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.

Long Noncoding RNA Expression during Human B-Cell Development.

Long noncoding RNAs (lncRNAs) have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre-B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n = 7), and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n = 6), respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 antisense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we analysed their co-expression with well-characterized protein-coding genes, a method known as "guilt by association". By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignancies that originate from distinct B-cell subpopulations.

Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifs.

Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3'UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.

A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent.

Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Hepatotoxic potential of therapeutic oligonucleotides can be predicted from their sequence and modification pattern.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma.

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains elusive. Recent discoveries indicate that the oncogenic microRNA miR-155 is overexpressed in affected skin from CTCL patients. Here, we address what drives the expression of miR-155 and investigate its role in the pathogenesis of CTCL. We show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells. Incubation with JAK inhibitor or siRNA-mediated knockdown of STAT5 decreases BIC/miR-155 expression, whereas IL-2 and IL-15 increase their expression in cell lines and primary cells. In contrast, knockdown of STAT3 has no effect, and BIC is not a transcriptional target of STAT3, indicating that regulation of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC.   In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data indicate that the STAT5/BIC/miR-155 pathway promotes proliferation of malignant T cells, and therefore is a putative target for therapy in CTCL.

Divalent metal transporter 1 regulates iron-mediated ROS and pancreatic ß cell fate in response to cytokines.

Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1ß induces divalent metal transporter 1 (DMT1) expression correlating with increased ß cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, ß cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.

Allergic contact dermatitis induces upregulation of identical microRNAs in humans and mice.

BACKGROUND: MicroRNAs are short, endogenous RNA molecules that can bind to parts of target mRNAs, thus inhibiting their translation and causing accelerated turnover or degradation of transcripts, thereby regulating gene expression. Several microRNAs have been found to be upregulated in atopic dermatitis and psoriasis, indicating a role in inflammatory skin diseases. However, there have been no studies on the expression of microRNAs in allergic contact dermatitis. OBJECTIVES: To investigate expression of microRNAs in allergic contact dermatitis. Methods. Lesional and non-lesional skin biopsies were collected from subjects with allergic responses to diphenylcyclopropenone (DPCP). Additional samples for profiling were collected from an experimental mouse model by use of the strong allergen dinitrofluorobenzene. RNA was purified from all samples, and locked nucleic acid microarray analysis was performed, followed by validation with quantitative polymerase chain reaction (PCR). RESULTS: In humans sensitized with DPCP, we found significant upregulation of miR-21, miR-142-3p, miR-142-5p and miR-223 in challenged skin. The same microRNAs were significantly upregulated in the skin of mice in a mouse model of contact allergy. The upregulation of microRNA was confirmed by quantitative PCR. CONCLUSION: These are the first results indicating that microRNAs may be involved in the pathogenesis of allergic contact dermatitis, and they show that mouse models are valuable tools for further study of the involvement of microRNAs in allergic contact dermatitis.

Comparison of microRNA expression using different preservation methods of matched psoriatic skin samples.

MicroRNAs are non-coding RNA molecules modulating gene expression post-transcriptionally. Formalin-fixed, paraffin-embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high-quality RNA from human skin can be challenging as skin contains high levels of RNases. As microRNAs are 19-23 nucleotides long and lack a poly-A tail, they may be less prone to RNA degradation than mRNAs. We investigated whether microRNAs in psoriatic (FFPE) samples reliably reflect microRNA expression in samples less prone to RNA degradation such as fresh-frozen (FS) and Tissue-Tek-embedding (OCT). We found a strong correlation of the microRNA expression levels between all preservation methods of matched psoriatic skin samples (r(s) ranging from 0.91 to 0.95 (P < 0.001)). These observations were further confirmed with qRT-PCR. Our results demonstrate that microRNA detection in human skin is robust irrespective of preservation method; thus, microRNAs offer an appropriate and flexible approach in clinical practices and for diagnostic purposes in skin disorders.