Small Oligonucleotides Research Articles

Inhibition of Factor XI using a GalNAc-siRNA RBD5049: a novel antithrombotic drug with high potency and long duration

Abstract Introduction Bleeding risk remains a major concern of the existing anticoagulants. Inhibition Factor XI (FXI) using small molecule inhibitors, antibodies or antisense oligonucleotides (ASOs) to inactive intrinsic pathway of coagulation have been developed as a safer treatment option over the last decade. siRNA is a nucleic acid drug with class advantages of high selectivity, long duration and benign safety profiles. The current abstract summarizes key preclinical efficacy and safety data supporting the first-in-human study. Purpose To evaluate efficacy and safety, of a GalNAc-siRNA molecule RBD4059, for development of a novel antithrombotic treatment. Methods In vitro potency of several siRNAs were assessed and screened in HepG2 cells. Upon selection of the lead candidate, the siRNA was conjugated with a GalNAc molecule, using RIBO-GalSTAR liver targeting system. In vivo efficacy of RBD4059 was tested in C57BL/6J mice and cynomolgus non-human primates (NHPs). The antithrombotic effect of RBD4059 was determined in arterial and venous thrombosis models induced by FeCl3. Enoxaparin sodium (Lovenox) with clinical equivalent dose (4 mg/kg) was used as a control. Single- and repeated- dose GLP toxicology studies were performed in both CD-1 mice and NHPs. Results IC50 values of RBD4059 was 0.56 nM HepG2 cells. Dose-dependent and long-lasting effects of RBD4059 on the inhibition of plasma FXI activity (FXIa) were observed in mice and in NHPs. RBD4059 at a single dose of 9 mg/kg achieved a maximum of 89% plasma FXIa reduction and 1.38-fold APTT prolongation in mice, and the inhibitory effects could be maintained until Day 64. RBD4059 at 3 mg/kg or 9 mg/kg significantly prevented flow reduction due to FeCl3-induced thrombosis in both arterial and venous thrombosis models, and efficacy was superior than enoxaparin. In NHPs, the inhibitory effects of FXIa were maintained until Day 71 with the maximum effect of 77% FXIa reduction and 1.34-fold APTT prolongation. GLP toxicology studies showed good safety profile without any signs of increased bleeding despite supra-physiological doses and the NOAEL was considered 600 mg/kg in mice and 400 mg/kg in NHPs. Conclusions RBD4059 has demonstrated high potency/efficacy and long duration regarding FXI inhibition and with large safety margin. As the first siRNA based molecule targeting FXI, RBD4059 has now entered Phase 1 trial in human healthy volunteers.Figure 1

Glucagon receptor modulation: Its role in diabetes care

Abstract The metabolic changes attributable to diabetes are due to both deficiency of insulin as well as excess glucagon (GC). The glucagon receptor (GCGR) is a G protein-coupled receptor. GC is predominantly found in the liver. GC exerts its action both at the GCGR and the glucagon-like peptide receptor (GLP1R). Knockout studies as well as human mutation analysis have indicated therapeutic potential as well as concerns of modulation of the GCGR as a potential target for diabetes therapies. Three approaches to GCGR antagonism—small molecules, monoclonal antibodies, and antisense oligonucleotides—have demonstrated an impressive reduction in HbA1C in preclinical and clinical studies. However, adverse events such as an increase in weight cholesterol liver enzymes and alpha cell hyperplasia have stalled further clinical development. On the other hand, balancing the hyperglycemic effect of glucagon and retaining their beneficial effects, especially with weight loss and decrease in weight loss and hepatic steatosis by use of unimolecular coagonists with GLP1 and GIP have shown considerable promise in clinical trials.

Advancing Epidemiology and Genetic Approaches for the Treatment of Spinal and Bulbar Muscular Atrophy: Focus on Prevalence in the Indigenous Population of Western Canada.

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a debilitating neuromuscular disease characterized by progressive muscular weakness and neuronal degeneration, affecting 1-2 individuals per 100,000 globally. While SBMA is relatively rare, recent studies have shown a significantly higher prevalence of the disease among the indigenous population of Western Canada compared to the general population. The disease is caused by a pathogenic expansion of polyglutamine residues in the androgen receptor protein, which acts as a key transcriptional regulator for numerous genes. SBMA has no cure, and current treatments are primarily supportive and focused on symptom management. Recently, a form of precision medicine known as antisense therapy has gained traction as a promising therapeutic option for numerous neuromuscular diseases. Antisense therapy uses small synthetic oligonucleotides to confer therapeutic benefit by acting on pathogenic mRNA molecules, serving to either degrade pathogenic mRNA transcripts or helping to modulate splicing. Recent studies have explored the suitability of antisense therapy for the treatment of SBMA, primarily focused on gene therapy and antisense-mediated mRNA knockdown approaches. Advancements in understanding the pathogenesis of SBMA and the development of targeted therapies offer hope for improved quality of life for individuals affected by this debilitating condition. Continued research is essential to optimize these genetic approaches, ensuring their safety and efficacy.

Construction of DNA Bilayer Tiles and Arrays Using Circular DNA Molecules as Scaffolds.

Using oligonucleotides to weave 2D tiles such as double crossovers (DX) and multi-arm junction (mAJ) tiles and arrays is well-known, but weaving 3D tiles is rare. Here, we report the construction of two new bilayer tiles in high yield using small circular 84mer oligonucleotides as scaffolds. Further, we designed five E-tiling approaches to construct porous nanotubes of microns long in medium yield via solution assembly and densely covered planar microscale arrays via surface-mediated assembly.

Novel epigenetic molecular therapies for imprinting disorders.

Genomic imprinting disorders are caused by the disruption of genomic imprinting processes leading to a deficit or increase of an active allele. Their unique molecular mechanisms underlying imprinted genes offer an opportunity to investigate epigenetic-based therapy for reactivation of an inactive allele or reduction of an active allele. Current treatments are based on managing symptoms, not targeting the molecular mechanisms underlying imprinting disorders. Here, we highlight molecular approaches of therapeutic candidates in preclinical and clinical studies for individual imprinting disorders. These include the significant progress of discovery and testing of small molecules, antisense oligonucleotides, and CRISPR mediated genome editing approaches as new therapeutic strategies. We discuss the significant challenges of translating these promising therapies from the preclinical stage to the clinic, especially for genome editing based approaches.

Alpha-Gal Bound Aptamer and Vancomycin Synergistically Reduce Staphylococcus aureus Infection In Vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pervasive and persistent threat that requires the development of novel therapies or adjuvants for existing ones. Aptamers, small single-stranded oligonucleotides that form 3D structures and can bind to target molecules, provide one possible therapeutic route, especially when presented in combination with current antibiotic applications. BALB/c α-1, 3-galactosyltransferase (-/-) knockout (GTKO) mice were infected with MRSA via tail vein IV and subsequently treated with the αSA31 aptamer (n = 4), vancomycin (n = 12), or αSA31 plus vancomycin (n = 12), with split doses in the morning and evening. The heart, lungs, liver, spleen, and kidneys were harvested upon necropsy for histological and qPCR analysis. All mice treated with αSA31 alone died, whereas 5/12 mice treated with vancomycin alone and 7/12 mice treated with vancomycin plus αSA31 survived the course of the experiment. The treatment of MRSA-infected mice with Vancomycin and an adjuvant aptamer αSA31 reduced disease persistence and dispersion as compared to treatment with either vancomycin SA31 alone, indicating the combination of antibiotic and specifically targeted αSA31 aptamer could be a novel way to control MRSA infection. The data further indicate that aptamers may serve as a potential therapeutic option for other emerging antibiotic resistant pathogens.

Development of an electrochemiluminescent oligonucleotide-based assay for the quantification of prostate cancer associated miR-141-3p in human serum

MicroRNAs (miRNAs) are small oligonucleotides (18–25 bases), biologically relevant for epigenetic regulation of key processes, particularly in association with cancer. Research effort has therefore been directed towards the monitoring and detection of miRNAs to progress (early) cancer diagnoses. Traditional detection strategies for miRNAs are expensive, with a lengthy time-to-result. In this study we develop an oligonucleotide-based assay using electrochemistry for the specific, selective and sensitive detection of a circulating miRNA (miR-141) associated with prostate cancer. In the assay, the excitation and readout of the signal are independent: an electrochemical stimulation followed by an optical readout. A ‘sandwich’ approach is incorporated, consisting of a biotinylated capture probe immobilised on streptavidin-functionalised surfaces and a detection probe labelled with digoxigenin. We show that the assay allows the detection of miR-141 in human serum, even in the presence of other miRNAs, with a LOD of 0.25 pM. The developed electrochemiluminescent assay has, therefore, the potential for efficient universal oligonucleotide target detection via the redesign of capture and detection probes.

Splicing activates transcription from weak promoters upstream of alternative exons

Transcription and splicing are intrinsically coupled. Alternative splicing of internal exons can fine-tune gene expression through a recently described phenomenon called exon-mediated activation of transcription starts (EMATS). However, the association of this phenomenon with human diseases remains unknown. Here, we develop a strategy to activate gene expression through EMATS and demonstrate its potential for treatment of genetic diseases caused by loss of expression of essential genes. We first identified a catalog of human EMATS genes and provide a list of their pathological variants. To test if EMATS can be used to activate gene expression, we constructed stable cell lines expressing a splicing reporter based on the alternative splicing of motor neuron 2 (SMN2) gene. Using small molecules and antisense oligonucleotides (ASOs) currently used for treatment of spinal muscular atrophy, we demonstrated that increase of inclusion of alternative exons can trigger an activation of gene expression up to 45-fold by enhancing transcription in EMATS-like genes. We observed the strongest effects in genes under the regulation of weak human promoters located proximal to highly included skipped exons.

Design and development of microformulations for rapid release of small molecules and oligonucleotides

A systemic delivery of therapeutics frequently results in sub-optimal exposure of the targeted locus and undesired side effects. To address these challenges, a platform for local delivery of diverse therapeutics by remotely controlled magnetic micro-robots was introduced. The approach involves micro-formulation of active molecules using hydrogels that exhibit wide range of loading capabilities and predictable release kinetics. This work introduces two specific hydrogels based on thiol-maleimide and PEG-PLA-diacrylate chemistries that afford high, reliable and reproducible loading and release of several model molecules including doxorubicin, 25-mer poly-dT oligonucleotide and a 5.4 kBp GFP DNA plasmid. The described formulations are suitable for micro-dosing using both conventional or remote delivery devices.

Molecular contacts in the Cren7-DNA complex: A quantitative investigation for electrostatic interaction

The molecular association of proteins with nucleic acids leading to the formation of macromolecular complexes is a crucial step in several biological processes. Stabilization of these complexes involves electrostatic interactions between ion pairs (salt bridges) of nucleic acid phosphates and protein side chains. The crenarchaeal DNA binding protein, Cren7 plays a key role in the regulation of chromosomal structure and gene expression in eukaryotic extremophiles. However, the molecular contacts that occur at the interface of protein-DNA complexes and their contribution to the electrostatic interaction have not been fully elucidated. This work presents a quantitative description of the mechanism of the electrostatic interaction between the protein and DNA. We have identified a few residues located at the Cren7-DNA interface that could potentially be responsible for the interaction. Structural studies using circular dichroism indicate mutation of these surface residues minimally affect their structure and stability. The binding affinity of these mutants for the DNA duplexes was examined from reverse titration, biolayer interferometry, and fluorescence anisotropy measurements with calf thymus DNA, polynucleotides, and small DNA oligonucleotides. The resulting kinetic parameters highlight a difference in electrostatic interactions potentials exhibited by residues positioned at different locations of the protein-DNA interface. Computational studies attribute this difference to their surrounding atmosphere and energetic stabilization parameters. The biophysical approach described here can be extended for other proteins that play a crucial role in DNA bending and compaction, to properly evaluate the role of specific residues on the mechanisms of DNA binding.

Use of Microfluidics to Prepare Lipid-Based Nanocarriers.

Lipid-based nanoparticles (LBNPs) are an important tool for the delivery of a diverse set of drug cargoes, including small molecules, oligonucleotides, and proteins and peptides. Despite their development over the past several decades, this technology is still hindered by issues with the manufacturing processes leading to high polydispersity, batch-to-batch and operator-dependent variability, and limits to the production volumes. To overcome these issues, the use of microfluidic techniques in the production of LBNPs has sharply increased over the past two years. Microfluidics overcomes many of the pitfalls seen with conventional production methods, leading to reproducible LBNPs at lower costs and higher yields. In this review, the use of microfluidics in the preparation of various types of LBNPs, including liposomes, lipid nanoparticles, and solid lipid nanoparticles for the delivery of small molecules, oligonucleotides, and peptide/protein drugs is summarized. Various microfluidic parameters, as well as their effects on the physicochemical properties of LBNPs, are also discussed.

Measuring hybridization kinetics in live cells using a single-molecule imaging and tracking method.

The performance of many biosensors relies on effective hybridization of the nucleic acid probe with its complementary target, but the binding kinetics of probe-target pairs are often difficult to predict or measure in live cells. Current methods for studying annealing/melting kinetics of nucleic acids (NAs) mostly rely on in-vitro models that do not reflect the molecular constituents of native cellular environment. Here by using a transmembrane receptor labeled with SNAPf-tag and a DNA probe dually conjugated with benzylguanine (BG) and fluorophore Atto633 at the two ends, we measured the annealing/melting kinetics of a probe-target model system in live cells on a microscope. Upon electroporation, the BG-DNA-Atto633 probes were covalently linked to the SNAPf tags on receptors, thus anchored on the intracellular surfaces of plasma membrane. With Cy3-labeled complementary strands also in the cytoplasm, transient nucleic acid hybridization and melting between the two strands were observed through colocalization analysis at the single-molecule level. kon, koff and Kd were derived from the fluorescence time traces of Atto633 and Cy3 using the hidden Markov model (HMM). Although the observation depth (within 200 nm from the cell membrane) is much limited compared to that of the 3D tracking techniques (30 μm), hundreds of molecules can be tracked simultaneously and the resulting track durations are extended from a few hundreds of milliseconds to a few seconds, thus leading to more precise kinetics measurements. Our method provides a simple, direct and precise way to evaluate nucleic acid binding kinetics in cytoplasm. The acquired kinetics information is important for designing small nucleic acid probes or oligonucleotide drugs that bind with their specific targets effectively in live cells.

Targeting splicing factors for cancer therapy.

Alternative splicing (AS) of mRNAs is an essential regulatory mechanism in eukaryotic gene expression. AS misregulation, caused by either dysregulation or mutation of splicing factors, has been shown to be involved in cancer development and progression, making splicing factors suitable targets for cancer therapy. In recent years, various types of pharmacological modulators, such as small molecules and oligonucleotides, targeting distinct components of the splicing machinery, have been under development to treat multiple disorders. Although these approaches have promise, targeting the core spliceosome components disrupts the early stages of spliceosome assembly and can lead to nonspecific and toxic effects. New research directions have been focused on targeting specific splicing factors for a more precise effect. In this Perspective, we will highlight several approaches for targeting splicing factors and their functions and suggest ways to improve their specificity.

What Can RNA-Based Therapy Do for Monogenic Diseases?

The use of RNA-based approaches to treat monogenic diseases (i.e., hereditary disorders caused by mutations in single genes) has been developed on different fronts. One approach uses small antisense oligonucleotides (ASOs) to modulate RNA processing at various stages; namely, to enhance correct splicing, to stimulate exon skipping (to exclude premature termination codon variants), to avoid undesired messenger RNA (mRNA) transcript degradation via the nonsense-mediated decay (NMD) pathway, or to induce mRNA degradation where they encode toxic proteins (e.g., in dominant diseases). Another approach consists in administering mRNA, which, like gene therapy, is a mutation-agnostic approach with potential application to any recessive monogenic disease. This is simpler than gene therapy because instead of requiring targeting of the nucleus, the mRNA only needs to be delivered to the cytoplasm. Although very promising (as demonstrated by COVID-19 vaccines), these approaches still have potential for optimisation, namely regarding delivery efficiency, adverse drug reactions and toxicity.

Recent Advances on Small-Molecule Antagonists Targeting TLR7.

Toll-like receptor 7 (TLR7) is a class of pattern recognition receptors (PRRs) recognizing the pathogen-associated elements and damage and as such is a major player in the innate immune system. TLR7 triggers the release of pro-inflammatory cytokines or type-I interferons (IFN), which is essential for immunoregulation. Increasing reports also highlight that the abnormal activation of endosomal TLR7 is implicated in various immune-related diseases, carcinogenesis as well as the proliferation of human immunodeficiency virus (HIV). Hence, the design and development of potent and selective TLR7 antagonists based on small molecules or oligonucleotides may offer new tools for the prevention and management of such diseases. In this review, we offer an updated overview of the main structural features and therapeutic potential of small-molecule antagonists of TLR7. Various heterocyclic scaffolds targeting TLR7 binding sites are presented: pyrazoloquinoxaline, quinazoline, purine, imidazopyridine, pyridone, benzanilide, pyrazolopyrimidine/pyridine, benzoxazole, indazole, indole, and quinoline. Additionally, their structure-activity relationships (SAR) studies associated with biological activities and protein binding modes are introduced.

Gene therapy and its applications

Gene therapy is the treatment of abnormal or mutated genes present in cells through the addition of healthy genes or replacement/deletion/site-specific modification of faulty genes. Deoxyribonucleic acid, messenger ribonucleic acid (RNA), small interference RNA, microsomal RNA and antisense oligonucleotides are the genetic materials implicated in gene therapy. They are inserted into the diseased cells using viral or non-viral vectors through an in vivo or ex vivo transduction. Gamma retrovirus, lentivirus, herpesvirus, adenovirus and adeno-associated virus are common viral vectors, while transposons, cationic polymers, dendrimers and cell-penetrating peptides or liposomes are common non-viral vectors. Allologous or autologous T cells, haematopoietic stem cells and chimeric antigen receptor T cells are used for ex vivo gene transduction. Conventional gene therapy of inserting new genetic material shows toxicity such as off-target effects, altered immune responses, inflammatory reactions and possible oncogenic transformation in the recipient. Newer gene editing techniques such as zinc-finger nuclease, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats allow the site-specific correction or control of expression of mutated genes present in cells. Until August 2020, 23 gene-based medicines received approval from drug regulatory agencies in various countries and 362 were in development. Single-gene disorders have shown encouraging results, but evidence of using gene therapy in polygenic and common age-related diseases is still required. Recently, the horizon of gene therapy widened to include COVID vaccines and as an adjunct to chemotherapy. If we could overcome its limitations such as immunogenicity, mutagenicity and high costs, gene therapy can be the medicine of the next generation.

Principles and functions of condensate modifying drugs.

Biomolecular condensates are compartmentalized communities of biomolecules, which unlike traditional organelles, are not enclosed by membranes. Condensates play roles in diverse cellular processes, are dysfunctional in many disease states, and are often enriched in classically "undruggable" targets. In this review, we provide an overview for how drugs can modulate condensate structure and function by phenotypically classifying them as dissolvers (dissolve condensates), inducers (induce condensates), localizers (alter localization of the specific condensate community members) or morphers (alter the physiochemical properties). We discuss the growing list of bioactive molecules that function as condensate modifiers (c-mods), including small molecules, oligonucleotides, and peptides. We propose that understanding mechanisms of condensate perturbation of known c-mods will accelerate the discovery of a new class of therapies for difficult-to-treat diseases.

The Role of MicroRNAs in HER2-Positive Breast Cancer: Where We Are and Future Prospective.

Breast cancer that highly expresses human epidermal growth factor receptor 2 (HER2+) represents one of the major breast cancer subtypes, and was associated with a poor prognosis until the introduction of HER2-targeted therapies such as trastuzumab. Unfortunately, up to 30% of patients with HER2+ localized breast cancer continue to relapse, despite treatment. MicroRNAs (miRNAs) are small (approximately 20 nucleotides long) non-coding regulatory oligonucleotides. They function as post-transcriptional regulators of gene expression, binding complementarily to a target mRNA and leading to the arrest of translation or mRNA degradation. In the last two decades, translational research has focused on these small molecules because of their highly differentiated expression patterns in blood and tumor tissue, as well as their potential biological function. In cancer research, they have become pivotal for the thorough understanding of oncogenic biological processes. They might also provide an efficient approach to early monitoring of tumor progression or response to therapy. Indeed, changes in their expression patterns can represent a flag for deeper biological changes. In this review, we sum up the recent literature regarding miRNAs in HER2+ breast cancer, taking into account their potential as powerful prognostic and predictive biomarkers, as well as therapeutic tools.

Targeted co-delivery of a photosensitizer and an antisense oligonucleotide based on an activatable hyaluronic acid nanosystem with endogenous oxygen generation for enhanced photodynamic therapy of hypoxic tumors

Photodynamic therapy (PDT) is a promising cancer treatment modality with advantages of minimal invasiveness, repeatable therapy, and mild systemic toxicity. However, the limited bioavailability of photosensitizer (PS), tumor hypoxia, and the presence of antiapoptotic proteins in cancer cells, has hampered the efficiency of PDT. To address these limitations, herein, we developed a hyaluronic acid (HA) based nanosystem (HA-Ce6-Hemin@DNA-Protamine NPs, HCH@DP) loaded with chlorin e6 (Ce6, as PS), hemin (as mimetic catalase) and antisense oligonucleotide (ASO) of B-cell lymphoma 2 (Bcl-2) anti-apoptosis protein via a simple electrostatic self-assembly method for enhanced PDT of hypoxic solid tumors. The HCH@DP can target deliver the PS and ASO to tumor cells via cancer cell overexpressed HA receptors (i.e., CD44 or RHAMM). The Ce6 was released from HA-ss-Ce6 (HSC conjugates) after the reaction of cleavable disulfide bond with glutathione (GSH), which recovered the fluorescence and phototoxicity of Ce6 upon laser irradiation. Meanwhile, the catalase-mimicking hemin (degradation of HA-eda-hemin by hyaluronidase) decomposed the tumor overdressed endogenous H2O2 to oxygen, which relieved tumor hypoxia and further overcome hypoxia-associated resistance of PDT. Furthermore, the inhibition of Bcl-2 expression by Bcl-2 ASO also greatly improved the cellular sensitivity to PDT. Both in vitro and in vivo results showed the tumor cell targeting ability, hypoxia relief and significantly enhanced antitumor PDT efficacy of HCH@DP for hypoxic tumor cells upon laser irradiation. Thus, by improving the target delivery of PS and ASO, relieving tumor hypoxia, and down-regulation of anti-apoptotic proteins, this HCH@DP nanosystem achieved enhanced PDT efficiency against hypoxic tumors. In general, our work provided a promising strategy to increase the utilization of key components (PS and oxygen) of PDT and the cell sensitivity to PDT by targeting co-delivery PS and oligonucleotides to tumor cells via a biocompatible HA based carrier, thereby achieving efficiently PDT treatment of hypoxic solid tumors with potential translation possibility. Statement of significanceThe efficiency of PDT against solid tumor is severely restricted by the limited bioavailability of photosensitizer, tumor hypoxia, and the presence of antiapoptotic proteins in cancer cells. Herein, we have developed an activatable hyaluronic acid (HA) based nanosystem (HA-Ce6-Hemin@DNA-Protamine NPs, HCH@DP) via a simple electrostatic self-assembly method for PDT treatment of hypoxic solid tumors. The HCH@DP enabled to target co-delivery of photosensitizer and antisense oligonucleotide to tumor cells, overcoming tumor hypoxia through in situ oxygen production and improving cellular sensitivity by efficiently reducing anti-apoptosis effect of cancer cells for synergistically enhancing PDT efficiency. This work suggests a promising strategy to develop small molecule drug and oligonucleotides co-delivery nanoplatforms for efficiently PDT treatment of hypoxic solid tumor.

HER2 c-Terminal Fragments Are Expressed via Internal Translation of the HER2 mRNA.

The HER2/neu signaling pathway is one of the most frequently mutated in human cancer. Although therapeutics targeting this pathway have good efficacy, cancer cells frequently develop resistance. The HER2 gene encodes the full-length HER2 protein, as well as smaller c-terminal fragments (CTFs), which have been shown to be a cause of resistance. Here, we show that HER2 CTFs, exclusive from the full-length HER2 protein, are generated via internal translation of the full-length HER2 mRNA and identify regions which are required for this mechanism to occur. These regions of the HER2 mRNA may present novel sites for therapeutic intervention via small molecules or antisense oligonucleotides (ASOs).