Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Synthesis and evaluation of antisense oligonucleotides prodrug with G-quadruplex assembly and lysosome escape capabilities for oncotherapy.

The applications of antisense oligonucleotides (ASOs) in rare or common diseases treatment have garnered great attention in recent years. Nevertheless, challenges associated with stability and bioavailability still persist, hampering the efficiency of ASOs. This work presents an ASO prodrug with parallel G-quadruplex assembly and lysosome escape capabilities for oncotherapy. Our findings revealed that the end-assembled quadruplex structure effectively shielded the ASO from enzymatic degradation. Meanwhile, the conjugation of maleimide within the quadruplex enhanced cellular uptake, potentially offering an alternative cell entry mechanism that circumvents lysosome involvement. Notably, an optimized molecule, Mal2-G4-ASO, exhibited remarkable therapeutic effects both in vitro and in vivo. This work presents a promising avenue for enhancing the activity of nucleic acid drugs in oncotherapy and potentially other disease contexts.

Unveiling the potential of antisense oligonucleotides: Mechanisms, therapies, and safety insights.

Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded deoxynucleotide sequences composed of phosphate backbone-connected sugar rings. Designing of those strands is based on Watson-Crick hydrogen bonding mechanism. Thanks to rapidly advancing medicine and technology, evolving of the gene therapy area and ASO approaches gain attention. Considering the genetic basis of diseases, it is promising that gene therapy approaches offer more specific and effective options compared to conventional treatments. The objective of this review is to explain the mechanism of ASOs and discuss the characteristics and safety profiles of therapeutic agents in this field. Pharmacovigilance for gene therapy products is complex, requiring accurate assessment of benefit-risk balance and evaluation of adverse effects.

Coordination-Driven Self-Assembly of Metal Ion-Antisense Oligonucleotide Nanohybrids for Chronic Bacterial Infection Therapy.

Bacterial infection poses a significant challenge to wound healing and skin regeneration, leading to substantial economic burdens on patients and society. Therefore, it is crucial to promptly explore and develop effective methodologies for bacterial infections. Herein, we propose a novel approach for synthesizing nanostructures based on antisense oligonucleotides (ASOs) through the coordination-driven self-assembly of Zn2+ with ASO molecules. This approach aims to provide effective synergistic therapy for chronic wound infections caused by Staphylococcus aureus (S. aureus). The resulting hybrid nanoparticles successfully preserve the structural integrity and biological functionalities of ASOs, demonstrating excellent ASO encapsulation efficiency and bioaccessibility. In vitro antibacterial experiments reveal that Zn-ASO NPs exhibit antimicrobial properties against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. This antibacterial ability is attributed to the high concentration of metal zinc ions and the generation of high levels of reactive oxygen species. Additionally, the ftsZ-ASO effectively inhibits the expression of the ftsZ gene, further enhancing the antimicrobial effect. In vivo antibacterial assays demonstrate that the Zn-ASO NPs promote optimal skin wound healing and exhibit favorable biocompatibility against S. aureus infections, resulting in a residual infected area of less than 8%. This combined antibacterial strategy, which integrates antisense gene therapy and metal-coordination-directed self-assembly, not only achieves synergistic and augmented antibacterial outcomes but also expands the horizons of ASO coordination chemistry. Moreover, it addresses the gap in the antimicrobial application of metal-coordination ASO self-assembly, thereby advancing the field of ASO-based therapeutic approaches.

Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo.

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.

Light-Activated Nanodiamond-Based Drug Delivery Systems for Spatiotemporal Release of Antisense Oligonucleotides.

Nanodiamonds (NDs) are considered promising delivery platforms, but inaccurate and uncontrolled release of drugs at target sites is the biggest challenge of NDs in precision medicine. This study presents the development of phototriggerable ND-based drug delivery systems, utilizing ortho-nitrobenzyl (o-NB) molecules as photocleavable linkers between drugs and nanocarriers. UV irradiation specifically cleaved o-NB molecules and then was followed by releasing antisense oligonucleotides from ND-based carriers in both buffer and cellular environments. This ND system carried cell nonpermeable therapeutic agents for bypassing lysosomal trapping and degradation. The presence of fluorescent nitrogen-vacancy centers also allowed NDs to serve as biological probes for tracing in cells. We successfully demonstrated phototriggered release of antisense oligonucleotides from ND-based nanocarriers, reactivating their antisense functions. This highlights the potential of NDs, photocleavable linkers, and light stimuli to create advanced drug delivery systems for controlled drug release in disease therapy, opening possibilities for targeted and personalized treatments.

A comparative analysis of peptide-delivered antisense antibiotics using diverse nucleotide mimics.

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.

Types of RNA therapeutics.

RNA therapy is one of the new treatments using small RNA molecules to target and regulate gene expression. It involves the application of synthetic or modified RNA molecules to inhibit the expression of disease-causing genes specifically. In other words, it silences genes and suppresses the transcription process. The main theory behind RNA therapy is that RNA molecules can prevent the translation into proteins by binding to specific messenger RNA (mRNA) molecules. By targeting disease-related mRNA molecules, RNA therapy can effectively silence or reduce the development of harmful proteins. There are different types of RNA molecules used in therapy, including small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamer, ribozyme, and antisense oligonucleotides (ASOs). These molecules are designed to complement specific mRNA sequences, allowing them to bind and degrade the targeted mRNA or prevent its translation into protein. Nanotechnology is also highlighted to increase the efficacy of RNA-based drugs. In this chapter, while examining various methods of RNA therapy, we discuss the advantages and challenges of each.

Organ/Cell-Selective Intracellular Delivery of Biologics via N-Acetylated Galactosamine-Functionalized Polydisulfide Conjugates.

Biologics, including proteins and antisense oligonucleotides (ASOs), face significant challenges when it comes to achieving intracellular delivery within specific organs or cells through systemic administrations. In this study, we present a novel approach for delivering proteins and ASOs to liver cells, both in vitro and in vivo, using conjugates that tether N-acetylated galactosamine (GalNAc)-functionalized, cell-penetrating polydisulfides (PDSs). The method involves the thiol-bearing cargo-mediated ring-opening polymerization of GalNAc-functionalized lipoamide monomers through the so-called aggregation-induced polymerization, leading to the formation of site-specific protein/ASO-PDS conjugates with narrow dispersity. The hepatocyte-selective intracellular delivery of the conjugates arises from a combination of factors, including first GalNAc binding with ASGPR receptors on liver cells, leading to cell immobilization, and the subsequent thiol-disulfide exchange occurring on the cell surface, promoting internalization. Our findings emphasize the critical role of the close proximity of the PDS backbone to the cell surface, as it governs the success of thiol-disulfide exchange and, consequently, cell penetration. These conjugates hold tremendous potential in overcoming the various biological barriers encountered during systemic and cell-specific delivery of biomacromolecular cargos, opening up new avenues for the diagnosis and treatment of a range of liver-targeting diseases.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics.

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.

Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties.

Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.

Prodrug-Type Phosphotriester Oligonucleotides with Linear Disulfide Promoieties Responsive to Reducing Environment.

Various chemical modifications have been developed to create new antisense oligonucleotides (AONs) for clinical applications. Our previously designed prodrug-type phosphotriester-modified oligonucleotide with cyclic disulfides (cyclic SS PTE ON) can be converted into unmodified ON in an intracellular-mimetic reducing environment. However, the conversion rate of the cyclic SS PTE ON was very low, and the AON with cyclic SS PTE modifications showed much weaker antisense activity than corresponding to the fully phosphorothioate-modified AON. In this study, we synthesized several types of PTE ONs containing linear disulfides (linear SS PTE ONs) and evaluated their conversion rates under reducing conditions. From the results, the structural requirements for the conversion of the synthesized linear SS PTE ONs were elucidated. Linear SS PTE ON with promising promoieties showed a nuclease resistance up to 4.8-fold compared to unmodified ON and a cellular uptake by endocytosis without any transfection reagent. In addition, although the knockdown activity of the linear SS PTE gapmer AON is weaker than that of the fully phosphorothioate-modified gapmer AON, the knockdown activity is slightly stronger than that of the cyclic SS PTE gapmer AON. These results suggest that the conversion rates may be related to the expression of the antisense activity.

Post-Synthetic Nucleobase Modification of Oligodeoxynucleotides by Sonogashira Coupling and Influence of Alkynyl Modifications on the Duplex-Forming Ability.

Recently, it was reported that the alkynyl modification of nucleobases mitigates the toxicity of antisense oligonucleotides (ASO) while maintaining the efficacy. However, the general effect of alkynyl modifications on the duplex-forming ability of oligonucleotides (ONs) is unclear. In this study, post-synthetic nucleobase modification by Sonogashira coupling in aqueous medium was carried out to efficiently evaluate the physiological properties of various ONs with alkynyl-modified nucleobases. Although several undesired reactions, including nucleobase cyclization, were observed, various types of alkynyl-modified ONs were successfully obtained via Sonogashira coupling of ONs containing iodinated nucleobases. Evaluation of the stability of the duplex formed by the synthesized alkynyl-modified ONs showed that the alkynyl modification of pyrimidine was less tolerated than that of purine, although both the modifications occurred in the major groove of the duplex. These results can be attributed to the bond angle of the alkyne on the pyrimidine and the close proximity of the alkynyl substituents to the phosphodiester backbone. The synthetic method developed in this study may contribute to the screening of the optimal chemical modification of ASO because various alkynyl-modified ONs that are effective in reducing the toxicity of ASO can be easily synthesized by this method.

In Vivo Metabolite Profiles of an N-Acetylgalactosamine-Conjugated Antisense Oligonucleotide AZD8233 Using Liquid Chromatography High-Resolution Mass Spectrometry: A Cross-Species Comparison in Animals and Humans.

AZD8233, a liver-targeting antisense oligonucleotide (ASO), inhibits subtilisin/kexin type 9 protein synthesis. It is a phosphorothioated 3-10-3 gapmer with a central DNA sequence flanked by constrained 2'-O-ethyl 2',4'-bridged nucleic acid (cEt-BNA) wings and conjugated to a triantennary N-acetylgalactosamine (GalNAc) ligand at the 5'-end. Herein we report the biotransformation of AZD8233, as given by liver, kidney, plasma and urine samples, after repeated subcutaneous administration to humans, mice, rats, rabbits, and monkeys. Metabolite profiles were characterized using liquid chromatography high-resolution mass spectrometry. Metabolite formation was consistent across species, mainly comprising hydrolysis of GalNAc sugars, phosphodiester-linker hydrolysis releasing the full-length ASO, and endonuclease-mediated hydrolysis within the central DNA gap followed by exonuclease-mediated 5'- or 3'-degradation. All metabolites contained the 5'- or 3'-cEt-BNA terminus. Most shortmer metabolites had the free terminal alcohol at 5'- and 3'-positions of ribose, although six were found retaining the terminal 5'-phosphorothioate group. GalNAc conjugated shortmer metabolites were also observed in urine. Synthesized metabolite standards were applied for (semi)quantitative metabolite assessment. Intact AZD8233 was the major component in plasma, whereas the unconjugated full-length ASO was predominant in tissues. In plasma, most metabolites were shortmers retaining the 3'-cEt-BNA terminus, whereas metabolites containing the 5'- or 3'-cEt-BNA terminus were detected in both tissues and urine. All metabolites in human plasma were also detected in all nonclinical species, and all human urine metabolites were detected in monkey urine. In general, metabolite profiles in animal species were qualitatively similar and quantitatively exceeded the exposures of the circulating metabolites in humans at the doses studied. SIGNIFICANCE STATEMENT: This study presents metabolite identification and profiling of AZD8233, an N-acetylgalactosamine-conjugated antisense oligonucleotide (ASO), across species. A biotransformation strategy for ASOs was established by utilizing biologic samples collected from toxicology and/or clinical studies and liquid chromatography high-resolution mass spectrometry analysis without conducting bespoke radiolabeled absorption, distribution, metabolism, and excretion studies. The generated biotransformation package was considered adequate by health authorities to progress AZD8233 into a phase 3 program, proving its applicability to future metabolism studies of ASOs in drug development.

Mechanism of the extremely high duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine- or N-tert-butyl-N'- methylguanidine-bridged nucleic acids.

Antisense oligonucleotides (ASOs) are becoming a promising class of drugs for treating various diseases. Over the past few decades, many modified nucleic acids have been developed for application to ASOs, aiming to enhance their duplex-forming ability toward cognate mRNA and improve their stability against enzymatic degradations. Modulating the sugar conformation of nucleic acids by substituting an electron-withdrawing group at the 2'-position or incorporating a 2',4'-bridging structure is a common approach for enhancing duplex-forming ability. Here, we report on incorporating an N-tert-butylguanidinium group at the 2',4'-bridging structure, which greatly enhances duplex-forming ability because of its interactions with the minor groove. Our results indicated that hydrophobic substituents fitting the grooves of duplexes also have great potential to increase duplex-forming ability.

Non-Invasive Transdermal Delivery of Antisense Oligonucleotides with Biocompatible Ionic Liquids.

Nucleic acid drugs, including antisense oligonucleotides (ASOs), have received considerable attention as novel therapeutics for the treatment of intractable diseases. Despite their potential benefits, ASOs are currently administered via injection, which can negatively impact patient quality of life because of the prevalence of severe injection site reactions. Non-invasive transdermal administration of ASOs is desirable but highly challenging owing to the strong barrier imposed by the stratum corneum, which only permits the penetration of small molecules under 500 Da. For ASOs to exert their antisense effect, they must traverse the negatively charged cell membrane and reach the cytoplasm. In this study, we used the solid-in-oil (S/O) dispersion technology to facilitate the skin permeation of ASOs by coating the drug with a hydrophobic surfactant molecule, specifically lipid-based ionic liquid (IL) surfactants with high biocompatibility and transdermal penetration-enhancing properties. To induce the antisense effect, it was important to achieve simultaneous transdermal delivery and intracellular entrapment of ASOs. In vitro investigations indicated that the newly prepared IL-S/O enhanced the transdermal penetration and intracellular delivery of ASOs, thus inhibiting mRNA translation of the target TGF-ß. In addition, in vivo investigations of tumor-bearing mice suggested that the anti-tumor effect of the IL-S/O was similar to that of injection. This study demonstrates the potential of non-invasive transdermal delivery carriers based on biocompatible ILs, which can be applied to a variety of nucleic acid drugs.

Alteration of target cleavage patterns and off-target reduction of antisense oligonucleotides incorporating 2-N-carbamoyl- or (2-pyridyl)guanine.

Antisense oligonucleotides (ASOs) are therapeutic modalities that are successfully used as pharmaceuticals. However, there remains a concern that treatment with ASOs may cleave mismatched RNAs other than the target gene, leading to numerous alterations in gene expression. Therefore, improving the selectivity of ASOs is of paramount importance. Our group has focused on the fact that guanine forms stable mismatched base pairs and has developed guanine derivatives with modifications at the 2-amino group, which potentially change the mismatch recognition ability of guanine and the interaction between ASO and RNase H. In this study, we evaluated the properties of ASOs containing two guanine derivatives, 2-N-carbamoyl-guanine and 2-N-(2-pyridyl)guanine. We conducted ultraviolet (UV) melting experiments, RNase H cleavage assays, in vitro knockdown assays, and off-target transcriptome analyses using DNA microarrays. Our results indicate that the target cleavage pattern of RNase H was altered by the modification with guanine. Furthermore, global transcript alteration was suppressed in ASO incorporating 2-N-(2-pyridyl)guanine, despite a decrease in the thermal mismatch discrimination ability. These findings suggest that chemical modifications of the guanine 2-amino group have the potential to suppress hybridization-dependent off-target effects and improve ASO selectivity.

Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding.

We conducted a biophysical study to investigate the self-assembling and albumin-binding propensities of a series of fatty acid-modified locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers specific to the MALAT1 gene. To this end, a series of biophysical techniques were applied using label-free ASOs that were covalently modified with saturated fatty acids (FAs) of varying length, branching, and 5'/3' attachment. Using analytical ultracentrifugation (AUC), we demonstrate that ASOs conjugated with fatty acids longer than C16 exhibit an increasing tendency to form self-assembled vesicular structures. The C16 to C24 conjugates interacted via the fatty acid chains with mouse and human serum albumin (MSA/HSA) to form stable adducts with near-linear correlation between FA-ASO hydrophobicity and binding strength to mouse albumin. This was not observed for the longer fatty acid chain ASO conjugates (>C24) under the experimental conditions applied. The longer FA-ASO however adopted self-assembled structures with increasing intrinsic stabilities proportional to the fatty acid chain length. For instance, FA chain lengths smaller than C24 readily formed self-assembled structures containing 2 (C16), 6 (C22, bis-C12), and 12 (C24) monomers, as measured by analytical ultracentrifugation (AUC). Incubation with albumin disrupted these supramolecular architectures to form FA-ASO/albumin complexes mostly with 2:1 stoichiometry and binding affinities in the low micromolar range, as determined by isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC). Binding of FA-ASOs underwent a biphasic pattern for medium-length FA chain lengths (>C16) with an initial endothermic phase of particulate disruption, followed by an exothermic binding event to the albumin. Conversely, ASO modified with di-palmitic acid (C32) formed a strong, hexameric complex. This structure was not disrupted when incubated with albumin under conditions above the critical nanoparticle concentration (CNC; <0.4 µM). It is noteworthy that the interaction of parent, fatty acid-free malat1 ASO to albumin was below detectability by ITC (KD ≫150 µM). This work demonstrates that the nature of mono- vs multimeric structures of hydrophobically modified ASOs is governed by the hydrophobic effect. Consequently, supramolecular assembly to form particulate structures is a direct consequence of the fatty acid chain length. This provides opportunities to exploit the concept of hydrophobic modification to influence pharmacokinetics (PK) and biodistribution for ASOs in two ways: (1) binding of the FA-ASO to albumin as a carrier vehicle and (2) self-assembly resulting in albumin-inert, supramolecular architectures. Both concepts create opportunities to influence biodistribution, receptor interaction, uptake mechanism, and pharmacokinetics/pharmacodynamics (PK/PD) properties in vivo, potentially enabling access to extrahepatic tissues in sufficient concentration to treat disease.

Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides.

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.