Therapeutic Oligonucleotides Research Articles

Therapeutic-oligonucleotides activated by nucleases (TOUCAN): A nanocarrier system for the specific delivery of clinical nucleoside analogues

Nucleoside analogues have been in clinical use since 1960s and they are still used as the first therapeutic option for several cancers and viral infections, due to their high therapeutic efficacy. However, their wide clinical acceptance has been limited due to their high toxicity and severe side effects to patients. Herein, we report on a nanocarrier system that delivers nucleosides analogues in a target-specific manner, making nucleoside-based therapeutics safer and with the possibility to be used in other human conditions. This system, named, Therapeutic OligonUCleotides Activated by Nucleases” (TOUCAN) combines: i) the recognition power of oligonucleotides as substrates, ii) the use of nucleases as enzymatic biomarkers and iii) the clinical efficacy of nucleoside analogues, in a single approach. As a proof-of-concept, we report on a TOUCAN that is activated by a specific nuclease produced by bacteria and releases a therapeutic nucleoside, floxuridine. We demonstrate, for the first time, that, by incorporating a therapeutic nucleoside analogue into oligonucleotide probes, we can specifically inhibit bacterial growth in cultures. In this study, Staphylococcus aureus was selected as the targeted bacteria and the TOUCAN strategy successfully inhibited its growth with minimal inhibitory concentration (MIC) values ranging from 0.62 to 40 mg/L across all tested strains. Moreover, our results indicate that the intravenous administration of TOUCANs at a dose of 20 mg/kg over a 24-h period is a highly effective method for treating bacterial infections in a mouse model of pyomyositis. Importantly, no signs of toxicity were observed in our in vitro and in vivo studies. This work can significantly impact the current management of bacterial infections, laying the grounds for the development of a different class of antibiotics. Furthermore, it can provide a safer delivery platform for clinical nucleoside therapeutics in any human conditions, such as cancer and viral infection, where specific nuclease activity has been reported.

Developing oligonucleotide therapeutics with confidence

Developing oligonucleotide therapeutics with confidence

Solidification and oral delivery of biologics to the colon- A review

The oral delivery of biologics such as therapeutic proteins, peptides and oligonucleotides for the treatment of colon related diseases has been the focus of increasing attention over the last years. However, the major disadvantage of these macromolecules is their degradation propensity in liquid state which can lead to the undesirable and complete loss of function. Therefore, to increase the stability of the biologic and reduce their degradation propensity, formulation techniques such as solidification can be performed to obtain a stable solid dosage form for oral administration. Due to their fragility, stress exerted on the biologic during solidification has to be reduced with the incorporation of stabilizing excipients into the formulation. This review focuses on the state-of-the-art solidification techniques required to obtain a solid dosage form for the oral delivery of biologics to the colon and the use of suitable excipients for adequate stabilization upon solidification. The solidifying processes discussed within this review are spray drying, freeze drying, bead coating and also other techniques such as spray freeze drying, electro spraying, vacuum- and supercritical fluid drying. Further, the colon as site of absorption in both healthy and diseased state is critically reviewed and possible oral delivery systems for biologics are discussed.

Factors Influencing ADME Properties of Therapeutic Antisense Oligonucleotides: Physicochemical Characteristics and Beyond.

Therapeutic antisense oligonucleotides (ASOs) represent a diverse array of chemically modified singlestranded deoxyribonucleotides that work complementarily to affect their mRNA targets. They vastly differ from conventional small molecules. These newly developed therapeutic ASOs possess unique absorption, distribution, metabolism, and excretion (ADME) processes that ultimately determine their pharmacokinetic, efficacy and safety profiles. The ADME properties of ASOs and associated key factors have not been fully investigated. Therefore, thorough characterization and in-depth study of their ADME properties are critical to support drug discovery and development processes for safe and effective therapeutic ASOs. In this review, we discussed the main factors affecting the ADME characteristics of these novels and evolving therapies. The major changes to ASO backbone and sugar chemistry, conjugation approaches, sites and routes of administration, etc., are the principal determinants of ADME and PK profiles that consequentially impact their efficacy and safety profiles. In addition, species difference and DDI considerations are important in understanding ADME profile and PK translatability but are less studied for ASOs. We, therefore, have summarized these aspects based on current knowledge and provided discussions in this review. We also give an overview of the current tools, technologies, and approaches available to investigate key factors that influence the ADME of ASO drugs and provide future perspectives and knowledge gap analysis.

Optimization of orthogonal separations for the analysis of oligonucleotides using 2D-LC

Oligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO4) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods.

Synthesis and Structural Analysis of N3‐Methyluridine and 2’‐Alkoxy/Fluoro‐N3‐Methyluridine Nucleosides by Using NMR Spectroscopy, X‐Ray Crystallography, and Computational Methods

AbstractDual modifications (base and sugar) in siRNA have the potential to overcome off‐target effects and impart nuclease resistance. Here we present the synthesis and detailed structural analysis of N3‐methyluridine and 2’‐alkoxy/fluoro‐N3‐methyluridine modifications, the rationally designed modifications for oligonucleotide therapeutics. Using X‐ray crystallography and 1H NMR spectral data, it is clear that the C2’‐endo conformation of N3‐methyluridine switches to the C3’‐endo pucker state preferentially due to the introduction of electron‐withdrawing 2’‐alkoxy or fluoro substituents. DFT study confirms that the modifications preferentially acquire the C3’‐endo conformation. NBO analysis indicates that in the presence of the 2’‐alkoxy/fluoro substituents, the C3’‐endo conformation is stabilized due to nO4’→σ*C1’‐N1 anomeric as well as σC3’‐H→σ*C2’‐R and σC1’‐C2’→σ*C3’‐OH hyperconjugation effects. The W−C hydrogen bond energies of the modified nucleosides were calculated using the DFT and SAPT methods at the modified base pair regions, and it was observed that one of the hydrogen bonds gets disrupted due to the presence of the N3‐methyl group, causing significant decline in hydrogen bond energy. These detailed structural studies will help to expand the scope of these destabilizing modifications in the rational design of RNAi‐based therapeutics.

An enzyme cascade enables production of therapeutic oligonucleotides in a single operation.

Therapeutic oligonucleotides have emerged as a powerful drug modality with the potential to treat a wide range of diseases; however, the rising number of therapies poses a manufacturing challenge. Existing synthetic methods use stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. We report a biocatalytic approach to efficiently produce oligonucleotides in a single operation where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks and aqueous conditions. We demonstrate the versatility of this methodology through the synthesis of clinically relevant oligonucleotide sequences containing diverse modifications.

Role of microRNAs in Metabolic Diseases

MicroRNAs regulate a number of processes by controlling the gene expression at the post-transcriptional level. These small, non-coding RNA molecules play an important role in predisposition to metabolic diseases by maintaining glucose, lipid, and cholesterol metabolism. miRNAs are synthesized as double-stranded RNA, the two strands are separated to produce functional single-stranded miRNAs. The miRNAs are part of a large silencing complex called RNA-induced silencing complex (RISC). miRNAs regulate adipogenesis, the accumulation and droplet size of triacylglycerols, and PPAR gene expression. Dysfunction or higher expression of certain miRNAs (e.g., miR-222) silences the glucose transporter mRNAs resulting in a decrease in the surface receptors for glucose uptake hence predisposing to diabetes. miR-222 has also been reported to increase insulin resistance. Certain miRNAs are upregulated in obese mice as well as obese humans including miR143-145, miR29a, etc. The most important regulatory miRNA for lipid metabolism is miR-122, in addition, miR-33a and miR-33b exert their effect through sterol regulatory element binding proteins. The naturally occurring miRNAs can be silenced by chemically modified anti-miRNA oligonucleotides (AMOs) to alter their cellular expression levels. Another therapeutic oligonucleotide type called miRNA sponges compete with miRNAs for binding sites on the target mRNAs. The nuclease-resistant locked nucleic acids (LNAs) can also reduce the expression of certain miRNAs and have been shown to reduce cholesterol levels in mice. In conclusion, miRNAs appear to be faithful therapeutic targets for altering the gene expression of many target genes involved in lipid and fat metabolism in the future.

Developmental Strategies and Method Validation of LC-MS-Based Bioanalytical Methods for Oligonucleotide Therapeutics

Developmental Strategies and Method Validation of LC-MS-Based Bioanalytical Methods for Oligonucleotide Therapeutics

Challenges of Impurities Analysis in Manufacturing Therapeutic Oligonucleotides

Challenges of Impurities Analysis in Manufacturing Therapeutic Oligonucleotides

Targeted tissue delivery of RNA therapeutics using antibody-oligonucleotide conjugates (AOCs).

Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody-oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species. In mice, αTfR1 AOCs achieved a > 15-fold higher concentration to muscle tissue than unconjugated siRNA. A single dose of an αTfR1 conjugated to an siRNA against Ssb mRNA produced > 75% Ssb mRNA reduction in mice and monkeys, and mRNA silencing was greatest in skeletal and cardiac (striated) muscle with minimal to no activity in other major organs. In mice the EC50 for Ssb mRNA reduction in skeletal muscle was >75-fold less than in systemic tissues. Oligonucleotides conjugated to control antibodies or cholesterol produced no mRNA reduction or were 10-fold less potent, respectively. Tissue PKPD of AOCs demonstrated mRNA silencing activity primarily driven by receptor-mediated delivery in striated muscle for siRNA oligonucleotides. In mice, we show that AOC-mediated delivery is operable across various oligonucleotide modalities. AOC PKPD properties translated to higher species, providing promise for a new class of oligonucleotide therapeutics.

A single-cell map of antisense oligonucleotide activity in the brain.

Antisense oligonucleotides (ASOs) dosed into cerebrospinal fluid (CSF) distribute broadly throughout the central nervous system (CNS). By modulating RNA, they hold the promise of targeting root molecular causes of disease and hold potential to treat myriad CNS disorders. Realization of this potential requires that ASOs must be active in the disease-relevant cells, and ideally, that monitorable biomarkers also reflect ASO activity in these cells. The biodistribution and activity of such centrally delivered ASOs have been deeply characterized in rodent and non-human primate (NHP) models, but usually only in bulk tissue, limiting our understanding of the distribution of ASO activity across individual cells and across diverse CNS cell types. Moreover, in human clinical trials, target engagement is usually monitorable only in a single compartment, CSF. We sought a deeper understanding of how individual cells and cell types contribute to bulk tissue signal in the CNS, and how these are linked to CSF biomarker outcomes. We employed single nucleus transcriptomics on tissue from mice treated with RNase H1 ASOs against Prnp and Malat1 and NHPs treated with an ASO against PRNP. Pharmacologic activity was observed in every cell type, though sometimes with substantial differences in magnitude. Single cell RNA count distributions implied target RNA suppression in every single sequenced cell, rather than intense knockdown in only some cells. Duration of action up to 12 weeks post-dose differed across cell types, being shorter in microglia than in neurons. Suppression in neurons was generally similar to, or more robust than, the bulk tissue. In macaques, PrP in CSF was lowered 40% in conjunction with PRNP knockdown across all cell types including neurons, arguing that a CSF biomarker readout is likely to reflect ASO pharmacodynamic effect in disease-relevant cells in a neuronal disorder. Our results provide a reference dataset for ASO activity distribution in the CNS and establish single nucleus sequencing as a method for evaluating cell type specificity of oligonucleotide therapeutics and other modalities.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery invivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

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.

Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.

Design of a Phase 1, Placebo-Controlled Study to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Single-Ascending Doses of PGN-EDODM1 in Adult Participants with Myotonic Dystrophy Type 1 (DM1) (P6-8.004)

<h3>Objective:</h3> The primary objective of this single-ascending dose (SAD) study is to evaluate the safety and tolerability of PGN-EDODM1 in adults living with DM1. Secondary and exploratory objectives include pharmacokinetics (PK), muscle distribution, pharmacodynamics (changes in splicing pattern of transcripts), and select functional outcome measures. <h3>Background:</h3> PepGen’s Enhanced Delivery Oligonucleotide (EDO) cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides. PGN-EDODM1 is being evaluated for the treatment of DM1. PGN-EDODM1 binds to the pathogenic CUG repeat expansion in <i>DMPK</i> mRNA and reduces sequestration of Muscleblind (MBNL) proteins through a steric blocking mechanism. Release of MBNL proteins is hypothesized to correct DM1 spliceopathy; the root cause of DM1. <h3>Design/Methods:</h3> Males and females 18–50 years of age, inclusive, with genetically confirmed diagnosis of DM1 will be eligible to participate, and will be randomized 3:1 (6 active, 2 placebo) to receive PGN-EDODM1 or placebo in each dose cohort. A muscle needle biopsy (tibialis anterior) will be performed at baseline and at Weeks 4 and 16 post-dosing for measurement of splicing of selected transcripts. An independent data safety and monitoring committee will oversee dose escalation. <h3>Results:</h3> The doses to be assessed will be based on data from the preclinical data and data from the Phase 1 healthy volunteer safety study of our lead EDO, PGN-EDO51. A plan for analysis of safety measures such as vital signs, electrocardiograms, clinical chemistry, urinalysis, and biomarkers will be presented, along with details on proposed functional endpoints and biomarkers to be explored in this study. <h3>Conclusions:</h3> This Phase 1 SAD clinical study will support continued development of PGN-EDODM1 for the treatment of DM1, and will be initiated in 2023. Participants will be invited to enroll in a planned multiple-ascending dose study (MAD) and a subsequent open-label extension study. <b>Disclosure:</b> Dr. Mellion has received personal compensation for serving as an employee of PepGen. Dr. Mellion has stock in PepGen. Dr. Larkindale has received personal compensation for serving as an employee of PepGen. Jennifer Cormier has nothing to disclose. Ms. Hand has received personal compensation for serving as an employee of 4D Molecular Therapeutics. Ms. Hand has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Fulcrum Therapeutics, Inc.. Ms. Hand has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for PepGen Inc. Ms. Hand has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Bluebird Bio. Ms. Vacca has received personal compensation for serving as an employee of PepGen Inc. Ms. Vacca has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for PepGen Inc. Ms. Vacca has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Agios Pharmaceuticals. Ms. Vacca has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Nocion Therapeutics. Ms. Vacca has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Relmada Therapeutics. Ms. Vacca has stock in PepGen Inc.. Pallavi Lonkar has nothing to disclose. Dr. Holland has received personal compensation for serving as an employee of PepGen Inc.. Dr. Holland has received intellectual property interests from a discovery or technology relating to health care. Mr. Garg has received personal compensation for serving as an employee of PepGen. Mr. Garg has stock in pepgen. Mr. Foy has received personal compensation for serving as an employee of PepGen Inc.. Dr. McArthur has received personal compensation for serving as an employee of PepGen . Dr. McArthur has stock in PepGen .

PGN-EDODM1: Preclinical Data Supporting the Development of an Enhanced Delivery Oligonucleotide (EDO) for the Treatment of Myotonic Dystrophy Type 1 (DM1) (S48.001)

<h3>Objective:</h3> In vitro and in vivo evaluation of pharmacological activity via measurement of splicing changes, nuclear RNA foci, and myotonia. <h3>Background:</h3> PepGen’s EDO cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides. PGN-EDODM1 is being evaluated for the treatment of DM1. PGN-EDODM1 binds to the pathogenic CUG repeat expansion in <i>DMPK</i> mRNA and reduces sequestration of Muscleblind (MBNL) proteins within nuclear RNA foci through a steric blocking mechanism. Release of MBNL proteins is hypothesized to correct DM1 spliceopathy; a central cause of DM1. <h3>Design/Methods:</h3> DM1 donor cells with 2,600 CTG repeats were treated with PGN-EDODM1 (1, 2, 5, 10, or 20 μM). Nuclear RNA foci profiles and splicing events were evaluated after 24 hours. PGN-EDODM1 (0, 10, 20, 30, and 50 mg/kg) was administered intravenously (IV) to wildtype (WT) or HSA^LR mice (DM1 murine model containing 250 CTG repeats in the <i>HSA</i> gene). Splicing profiles were evaluated in gastrocnemius and quadricep muscles 2-weeks post dose and myotonia was quantitatively assessed. Tissue levels of PGN-EDODM1 were analyzed. To explore the duration of pharmacological activity, PGN-EDODM1 (30 mg/kg IV) was administered to WT and HSA^LR mice and splicing profiles were evaluated 12 or 24 weeks post-dose. <h3>Results:</h3> The cellular model showed dose-dependent reduction in toxic RNA foci and correction of mis-splicing. In the HSA^LR model, a single dose resulted in high muscle concentrations of PGN-EDODM1, dose-dependent correction of mis-splicing (persisting over 24 weeks), and resolution of myotonia. PGN-EDODM1 was generally well tolerated at pharmacological doses. <h3>Conclusions:</h3> These preclinical studies demonstrate that PGN-EDODM1 reduced nuclear RNA foci and corrected mis-splicing at clinically relevant, tolerable doses with effects lasting up to 24 weeks. The HSA^LR mouse showed resolution of myotonia after a single PGN-EDODM1 dose. A Phase 1 clinical study in adults with DM1 will be initiated in 2023. <b>Disclosure:</b> Dr. Holland has received personal compensation for serving as an employee of PepGen Inc.. Dr. Holland has received intellectual property interests from a discovery or technology relating to health care. Mr. Klein has nothing to disclose. Caroline Godfrey has nothing to disclose. Dr. Svenstrup has received personal compensation for serving as an employee of PepGen, Inc. Dr. Svenstrup has stock in PepGen, Inc. Dr. Larkindale has received personal compensation for serving as an employee of PepGen. Dr. Bracegirdle has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for PepGen Inc. Dr. Furling has nothing to disclose. Dr. Goyal has received personal compensation for serving as an employee of Pepgen Inc.. Dr. Goyal has received personal compensation for serving as an employee of Wave Life Sciences. Dr. Goyal has received personal compensation in the range of $100,000-$499,999 for serving as an officer or member of the Board of Directors for Pepgen Inc.. Dr. Goyal has stock in PepGen Inc..

PGN-EDO51: Preclinical Data Supporting the Development of an Enhanced Delivery Oligonucleotide (EDO) for the Treatment of Duchenne Muscular Dystrophy (DMD) (P3-8.002)

<h3>Objective:</h3> Pharmacological evaluation of mPGN-EDO23 (murine analogue of PGN-EDO51) was performed via assessment of serum biomarkers of muscle damage, exon skipping, and dystrophin production in <i>mdx</i> mice. Evaluation of PGN-EDO51 in non-human primates (NHPs) consisted of tissue distribution and measurement of exon skipping. <h3>Background:</h3> PepGen’s EDO cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides. PGN-EDO51 is being evaluated for the treatment of DMD amenable to exon 51 skipping. <h3>Design/Methods:</h3> A single dose of mPGN-EDO23 (0, 30, or 60 mg/kg) was administered intravenously (IV) to <i>mdx</i> mice. Creatine kinase, exon skipping, and dystrophin production were measured. In NHPs, biodistribution and exon skipping were measured following single (20, 40, and 60 mg/kg) and repeat (10, 30, and 60 mg/kg) IV administration of PGN-EDO51. <h3>Results:</h3> Single doses of mPGN-EDO23 reduced levels of creatine kinase in a dose-dependent manner. mPGN-EDO23 induced 93.1%, 86.3%, 76.6%, and 62.3% exon skipping and resulted in the induction of 90.4%, 99.7%, 80.6%, and 25.7% of normal dystrophin levels in the biceps, quadriceps, diaphragm, and heart, respectively. Following a single PGN-EDO51 dose, broad biodistribution was observed in NHPs. Repeat administration of 30 mg/kg PGN-EDO51 resulted in high levels of exon 51 skipping (78% in biceps, 73% in quadriceps, 76% in diaphragm, and 24% in heart). Exon skipping was higher with PGN-EDO51 than R6G-PMO51 (the most clinically advanced PPMO [peptide phosphorodiamidate morpholino oligomer] for exon 51 skipping) and the skipped transcript accumulated over time with repeat dosing. PGN-EDO51 was generally well tolerated at clinically relevant doses. <h3>Conclusions:</h3> These data demonstrate that EDOs result in high levels of exon skipping (mPGN-EDO23 and PGN-EDO51) and dystrophin production (mPGN-EDO23) and were well tolerated at clinically relevant doses. A Phase 2, multiple-ascending dose study in patients with DMD amenable to exon 51 skipping is planned for 2023. <b>Disclosure:</b> Dr. Holland has received personal compensation for serving as an employee of PepGen Inc.. Dr. Holland has received intellectual property interests from a discovery or technology relating to health care. Caroline Godfrey has nothing to disclose. Pallavi Lonkar has nothing to disclose. Dr. Svenstrup has received personal compensation for serving as an employee of PepGen, Inc. Dr. Svenstrup has stock in PepGen, Inc. Dr. Larkindale has received personal compensation for serving as an employee of PepGen. Dr. McArthur has received personal compensation for serving as an employee of PepGen . Dr. McArthur has stock in PepGen . Dr. Bracegirdle has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for PepGen Inc. Dr. Goyal has received personal compensation for serving as an employee of Pepgen Inc.. Dr. Goyal has received personal compensation for serving as an employee of Wave Life Sciences. Dr. Goyal has received personal compensation in the range of $100,000-$499,999 for serving as an officer or member of the Board of Directors for Pepgen Inc.. Dr. Goyal has stock in PepGen Inc..

Synthesis and properties of a novel modified nucleic acid, 2′-N-methanesulfonyl-2′-amino-locked nucleic acid

2′-Amino-locked nucleic acid has a functionalizable nitrogen atom at the 2′-position of its furanose ring that can provide desired properties to a nucleic acid as a scaffold. In this study, we synthesized a novel nucleic acid, 2′-N-methanesulfonyl-2′-amino-locked nucleic acid (ALNA[Ms]) and conducted comparative studies on the physical and pharmacological properties of the ALNA[Ms] and on conventional nucleic acids, such as 2′-methylamino-LNA (ALNA[Me]), which is a classical 2′-amino-LNA derivative, and also on 2′,4′-BNA/LNA (LNA). ALNA[Ms] oligomers exhibited binding affinities for the complementary RNA strand that are similar to those of conventional nucleic acids. Four types of ALNA[Ms] nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. The knockdown abilities of Malat1 RNA using the Matat1 antisense oligonucleotide (ASO) containing ALNA[Ms] were higher than those of ALNA[Me] and were closer to those of LNA. Furthermore, the ASO containing ALNA[Ms] showed different tissue tropism from that containing LNA. ALNA[Ms] exhibited biological activities that were distinct from conventional constrained nucleic acids, suggesting the possibility that ALNA[Ms] can serve as novel modified nucleic acids in oligonucleotide therapeutics.

Strategy for Pre-Clinical Development of Active Targeting MicroRNA Oligonucleotide Therapeutics for Unmet Medical Needs.

We present here an innovative modular and outsourced model of drug research and development for microRNA oligonucleotide therapeutics (miRNA ONTs). This model is being implemented by a biotechnology company, namely AptamiR Therapeutics, in collaboration with Centers of Excellence in Academic Institutions. Our aim is to develop safe, effective and convenient active targeting miRNA ONT agents for the metabolic pandemic of obesity and metabolic-associated fatty liver disease (MAFLD), as well as deadly ovarian cancer.