Self-Assembled Antibody-Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides.

Antibody-oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO).  Here, we have developed a modular AOC (MAOC) approach based on accurate self-assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO-to-antibody ratio were generated by L-DNA scaffold mediated precise self-assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti-TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo-EM structure of the sdAb-TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic Duchenne muscular dystrophy (DMD) mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity-related morbidity or mortality. Collectively, our data suggest that the self-assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi-specific targeting and delivery.

Nucleic Acid Armor: Fortifying RNA Therapeutics through Delivery and Targeting Innovations for Immunotherapy.

RNA is a promising nucleic acid-based biomolecule for various treatments because of its high efficacy, low toxicity, and the tremendous availability of targeting sequences. Nevertheless, RNA shows instability and has a short half-life in physiological environments such as the bloodstream in the presence of RNAase. Therefore, developing reliable delivery strategies is important for targeting disease sites and maximizing the therapeutic effect of RNA drugs, particularly in the field of immunotherapy. In this mini-review, we highlight two major approaches: (1) delivery vehicles and (2) chemical modifications. Recent advances in delivery vehicles employ nanotechnologies such as lipid-based nanoparticles, viral vectors, and inorganic nanocarriers to precisely target specific cell types to facilitate RNA cellular entry. On the other hand, chemical modification utilizes the alteration of RNA structures via the addition of covalent bonds such as N-acetylgalactosamine or antibodies (antibody-oligonucleotide conjugates) to target specific receptors of cells. The pros and cons of these technologies are enlisted in this review. We aim to review nucleic acid drugs, their delivery systems, targeting strategies, and related chemical modifications. Finally, we express our perspective on the potential combination of RNA-based click chemistry with adoptive cell therapy (e.g., B cells or T cells) to address the issues of short duration and short half-life associated with antibody-oligonucleotide conjugate drugs.

Bioanalysis of free antisense oligonucleotide payload from antibody-oligonucleotide conjugate by hybridization LC-MS/MS.

Background: Antisense oligonucleotides (ASOs) have been conjugated to various moieties, such as peptides, antibodies or Fab regions of antibodies, to enhance their delivery to target tissues. The quantitation of free ASO (ASO payload) is critical to characterize its pharmacokinetics/pharmacodynamics (PK/PD) properties and biodistribution after delivery of the peptide/antibody/Fab ASO conjugates.Results: We developed a hybridization-based LC-MS/MS methodology for quantification of free ASO in tissues in the presence of Fab-ASO and ASO with linker (ASO-linker).Conclusion: The developed method was applied to measure accurately the free ASO concentrations in liver and gastrocnemius in mice that were dosed with Fab-ASO. This methodology has also been applied to free ASO bioanalysis for other antibody-ASO and Fab-ASO conjugates in various tissues and plasma/serum samples.

[Box: see text].

Preface for special issue: "Emerging strategies, technologies, and approaches for the next generation ADCs".

1. Antibody-drug conjugates (ADCs) represent an advanced category of biotherapeutic agents, typically consisting of an antibody bound to a biologically-active cytotoxic agent. Since the first ADC, MylotargTM, was approved in 2000, there have been fifteen ADCs sanctioned to date, with thirteen receiving approval from the FDA for the treatment of a variety of cancers, including blood malignancies and solid tumors.2. In this Special Issue of Xenobiotica focusing on ADCs, our goal is to compile a collection of papers, featuring both original research and review articles authored by specialists in academia and the pharmaceutical industry, to showcase some of the historical insights gained, current progress, and future prospects to enhance comprehension and tackle obstacles in the field of ADC development for cancer therapy.3. This special issue features articles that evaluate key components of ADC development, including payload design, innovative linker chemistries, and the use of new technologies for site-specific conjugations beyond traditional engineered cysteines. It also spotlights cutting-edge ADC structures like bispecific ADCs, dual-payload ADCs, targeted nanoparticles and antibody oligonucleotide conjugates (AOCs).4. Several other papers discuss bioanalytical and ADME strategies for ADCs as well. In addition, approaches to improve the translation of pharmacokinetics, safety, and therapeutic index (TI) of ADCs are presented.

Precision targeting in oncology: The future of conjugated drugs.

Coupled drugs, especially antibody-coupled drugs (ADCs), are a hot topic in oncology. As the development of ADCs has progressed, different coupling modes have emerged, inspired by their structural design have emerged. Technological advances have led to interweaving and collision of old and new concepts of coupled drugs, and have even challenged the concepts and techniques of coupled drugs at this stage. For example, antibody-oligonucleotide conjugates are a new class of chimeric biomolecules synthesized by coupling oligonucleotides with monoclonal antibodies through linkers, offering precise targeting and improved pharmacokinetic properties. This study aimed to elucidate the mechanism of action of coupled drugs and their current development status in antitumor therapy to provide better strategies for antitumor therapy.

A Catalytic Approach for the Synthesis of Peptide-Oligonucleotides Conjugates in Aqueous Solution or On-Column.

Peptide-oligonucleotide conjugates (POCs) are covalent architectures composed of a DNA or RNA molecules linked to a peptide. These constructs have found widespread applications ranging from hybrid nanomaterials to gene-targeted therapies. Considering the important role of POCs, a new catalytic approach for their preparation is reported here, that could be applied either on solid support in anhydrous media, or post-synthetically in aqueous buffer. Single amino acids, peptides and cell penetrating peptides (CPPs) were conjugated to various oligo(ribo)nucleotides with high conversions and good isolated yields. The applicability of the method was demonstrated on more than 35 examples including an analogue of a commercial therapeutic oligonucleotide. Other conjugation partners, such as deoxycholic acid and biotin were also successfully conjugated to oligonucleotides. To highlight the potential of this catalytic approach, these conditions have been applied to iterative processes, which is of high interest for the development of DNA-Encoded Libraries.

Bioanalytical approaches to support the development of antibody-oligonucleotide conjugate (AOC) therapeutic proteins.

RNA interference (RNAi) is a biological process that evolved to protect eukaryotic organisms from foreign genes delivered by viruses. This process has been adapted as a powerful tool to treat numerous diseases through the delivery of small-interfering RNAs (siRNAs) to target cells to alter aberrant gene expression.Antibody-oligonucleotide conjugates (AOCs) are monoclonal antibodies with complexed siRNA or antisense oligonucleotides (ASOs) that have emerged to address some of the challenges faced by naked or chemically conjugated siRNA, which include rapid clearance from systemic circulation and lack of selective delivery of siRNA to target cells.It is essential to characterise the ADME properties of an AOC during development to optimise distribution to target tissues, to minimise the impact of biotransformation on exposure, and to characterise the PK/PD relationship to guide translation. However, owing to the complexity of AOC structure, this presents significant bioanalytical challenges, and multiple bioanalytical measurements are required to investigate the pharmacokinetics and biotransformation of the antibody, linker, and siRNA payload.In this paper, we describe an analytical workflow that details in vivo characterisation of AOCs through measurement of their distinct molecular components to provide the basis for greater understanding of their ADME properties. Although the approaches herein can be applied to in vitro characterisation of AOCs, this paper will focus on in vivo applications. This workflow relies on high-resolution mass spectrometry as the principal means of detection and leverages chromatographic, affinity-based, and enzymatic sample preparation steps.

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking.

The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics.

Interrogating heterogeneity of cysteine-engineered antibody-drug conjugates and antibody-oligonucleotide conjugates by capillary zone electrophoresis-mass spectrometry.

Production of site-specific cysteine-engineered antibody-drug conjugates (ADCs) in mammalian cells may produce developability challenges, fragments, and heterogenous molecules, leading to potential product critical quality attributes in later development stages. Liquid phase chromatography with mass spectrometry (LC-MS) is widely used to evaluate antibody impurities and drug-to-antibody ratio, but faces challenges in analysis of fragment product variants of cysteine-engineered ADCs and oligonucleotide-to-antibody ratio (OAR) species of antibody-oligonucleotide conjugates (AOCs). Here, for the first time, we report novel capillary zone electrophoresis (CZE)-MS approaches to address the challenges above. CZE analysis of six ADCs made with different parent monoclonal antibodies (mAbs) and small molecule drug-linker payloads revealed that various fragment impurities, such as half mAbs with one/two drugs, light chains with one/two drugs, light chains with C-terminal cysteine truncation, heavy chain clippings, were well resolved from the main species. However, most of these fragments were coeluted or had signal suppression during LC-MS analysis. Furthermore, the method was optimized on both ionization and separation aspects to enable the characterization of two AOCs. The method successfully achieved baseline separation and accurate quantification of their OAR species, which were also highly challenging using conventional LC-MS methods. Finally, we compared the migration time and CZE separation profiles among ADCs and their parent mAbs, and found that properties of mAbs and linker payloads significantly influenced the separation of product variants by altering their size or charge. Our study showcases the good performance and broad applicability of CZE-MS techniques for monitoring the heterogeneity of cysteine-engineered ADCs and AOCs.

Lipid and Peptide-Oligonucleotide Conjugates for Therapeutic Purposes: From Simple Hybrids to Complex Multifunctional Assemblies.

Antisense and small interfering RNA (siRNA) oligonucleotides have been recognized as powerful therapeutic compounds for targeting mRNAs and inducing their degradation. However, a major obstacle is that unmodified oligonucleotides are not readily taken up into tissues and are susceptible to degradation by nucleases. For these reasons, the design and preparation of modified DNA/RNA derivatives with better stability and an ability to be produced at large scale with enhanced uptake properties is of vital importance to improve current limitations. In the present study, we review the conjugation of oligonucleotides with lipids and peptides in order to produce oligonucleotide conjugates for therapeutics aiming to develop novel compounds with favorable pharmacokinetics.

Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates.

Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5' or 3' end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.

Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals.

We describe methods to follow the fate of oligonucleotides after their injection into experimental animals. The quantitation in various tissues, blood or bone marrow cells is possible by chemical ligation PCR. This method works independently of chemical modifications of the oligonucleotide and/or its conjugations to lipid or peptide moieties. Moreover, metabolization intermediates can be detected by mass spectrometry. Together with a readout assay for the biochemical or physiological effects, which will differ, depending on the particular purpose of the oligonucleotide, these methods allow for a comprehensive understanding of oligonucleotide behavior in a living organism.

Properties of Parallel Tetramolecular G-Quadruplex Carrying N-Acetylgalactosamine as Potential Enhancer for Oligonucleotide Delivery to Hepatocytes.

The development of oligonucleotide conjugates for in vivo targeting is one of the most exciting areas for oligonucleotide therapeutics. A major breakthrough in this field was the development of multifunctional GalNAc-oligonucleotides with high affinity to asialoglycoprotein receptors (ASGPR) that directed therapeutic oligonucleotides to hepatocytes. In the present study, we explore the use of G-rich sequences functionalized with one unit of GalNAc at the 3'-end for the formation of tetrameric GalNAc nanostructures upon formation of a parallel G-quadruplex. These compounds are expected to facilitate the synthetic protocols by providing the multifunctionality needed for the binding to ASGPR. To this end, several G-rich oligonucleotides carrying a TGGGGGGT sequence at the 3'-end functionalized with one molecule of N-acetylgalactosamine (GalNAc) were synthesized together with appropriate control sequences. The formation of a self-assembled parallel G-quadruplex was confirmed through various biophysical techniques such as circular dichroism, nuclear magnetic resonance, polyacrylamide electrophoresis and denaturation curves. Binding experiments to ASGPR show that the size and the relative position of the therapeutic cargo are critical for the binding of these nanostructures. The biological properties of the resulting parallel G-quadruplex were evaluated demonstrating the absence of the toxicity in cell lines. The internalization preferences of GalNAc-quadruplexes to hepatic cells were also demonstrated as well as the enhancement of the luciferase inhibition using the luciferase assay in HepG2 cell lines versus HeLa cells. All together, we demonstrate that tetramerization of G-rich oligonucleotide is a novel and simple route to obtain the beneficial effects of multivalent N-acetylgalactosamine functionalization.

Bulge-Forming miRNases Cleave Oncogenic miRNAs at the Central Loop Region in a Sequence-Specific Manner.

The selective degradation of disease-associated microRNA is promising for the development of new therapeutic approaches. In this study, we engineered a series of bulge-loop-forming oligonucleotides conjugated with catalytic peptide [(LeuArg)2Gly]2 (BC-miRNases) capable of recognizing and destroying oncogenic miR-17 and miR-21. The principle behind the design of BC-miRNase is the cleavage of miRNA at a three-nucleotide bulge loop that forms in the central loop region, which is essential for the biological competence of miRNA. A thorough study of mono- and bis-BC-miRNases (containing one or two catalytic peptides, respectively) revealed that: (i) the sequence of miRNA bulge loops and neighbouring motifs are of fundamental importance for efficient miRNA cleavage (i.e., motifs containing repeating pyrimidine-A bonds are more susceptible to cleavage); (ii) the incorporation of the second catalytic peptide in the same molecular scaffold increases the potency of BC-miRNase, providing a complete degradation of miR-17 within 72 h; (iii) the synergetic co-operation of BC-miRNases with RNase H accelerates the rate of miRNA catalytic cleavage by both the conjugate and the enzyme. Such synergy allows the rapid destruction of constantly emerging miRNA to maintain sufficient knockdown and achieve a desired therapeutic effect.

Evaluating Efficacy of Peptide-Delivered Oligonucleotides Using the Severe Taiwanese SMA Mouse Model.

Oligonucleotides (ONs) are therapeutic macromolecules with great potential for the treatment of neurological conditions, including spinal muscular atrophy (SMA), a neurodegenerative disease. However, the neurovascular unit severely limits their distribution to the neural parenchyma of the brain and the spinal cord. Cell-penetrating peptides (CPPs) can be conjugated to oligonucleotides to increase their delivery across biological barriers. In this chapter, we describe the synthesis and conjugation of CPPs to oligonucleotides, and the use of a severe SMA mouse model to test in vivo the efficacy of CPP-delivered oligonucleotides, using ELISA, western blot, and TaqMan™ RT-qPCR assays.

Förster Resonance Energy Transfer Nanoplatform Based on Recognition-Induced Fusion/Fission of DNA Mixed Micelles for Nucleic Acid Sensing.

The dynamic nature of micellar nanostructures is employed to form a self-assembled Förster resonance energy transfer (FRET) nanoplatform for enhanced sensing of DNA. The platform consists of lipid oligonucleotide FRET probes incorporated into micellar scaffolds, where single recognition events result in fusion and fission of DNA mixed micelles, triggering the fluorescence response of multiple rather than a single FRET pair. In comparison to conventional FRET substrates where a single donor interacts with a single acceptor, the micellar multiplex FRET system showed ∼20- and ∼3-fold enhancements in the limit of detection and FRET efficiency, respectively. This supramolecular signal amplification approach could potentially be used to improve FRET-based diagnostic assays of nucleic acid and non-DNA based targets.

Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates.

A summary of the key technological advancements in the preparation of antibody-oligonucleotide conjugates (AOCs) and the distinct advantages and disadvantages of AOCs as novel therapeutics are presented. The merits and demerits of the different approaches to conjugating oligonucleotides to antibodies, antibody fragments or other proteins, mainly from the perspective of AOC purification and analytical characterizations, are assessed. The lessons learned from in vitro and in vivo studies, especially the findings related to silencing, trafficking, and cytotoxicity of the conjugates, are also summarized.

Expanding the Scope of the Cleavable N-(methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates.

Oligonucleotides modified by a 2'-deoxy-2'-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-ß-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a ß-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to ß-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T0.5 ca 7 h) to hydrazone-based structures.

Polyamine-Oligonucleotide Conjugates: 2'-OMe-Triazole-Linked 1,4,7,10-Tetraazacyclododecane and Intercalating Dyes and Their Effect on the Thermal Stability of DNA Duplexes.

Oligonucleotides with the sequences 5'-GTG AUPA TGC, 5'-GCA TAUP CAC and 5'-GUPG ATA UPGC, where UP is 2'-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide-alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection-deprotection strategy that allows efficient separation of the resulting polyamine-oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine-oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modified oligonucleotides. An additive and destabilizing effect of cyclen moieties attached to dsDNAs was observed. Tm measurements indicate that placing the hydrophobic dye opposite to the cyclen moiety can reduce its destabilizing effect and increase the thermal stability of the duplex. Interestingly, the cyclen-modified U showed significant selectivity for TT mismatch, which resulted in stabilization of the duplex. We conclude the paper with a brief review and discussion in which we compare our results with several examples of oligonucleotides labeled with polyamines at internal strand positions known in the literature.

Terminal Mono- and Bis-Conjugates of Oligonucleotides with Closo-Dodecaborate: Synthesis and Physico-Chemical Properties.

Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, the closo-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, whereas analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.