Antisense Oligonucleotide Sequences Research Articles

Antisense oligonucleotide as a new technology application for CsLOB1 gene silencing aiming citrus canker resistance.

Citrus canker disease, caused by Xanthomonas citri subsp. citri, poses a significant threat to global citrus production. The control of the disease in the field relies mainly on the use of conventional tools such as copper compounds, which are harmful to the environment and could lead to bacterial resistance. This scenario stresses the need for new and sustainable technologies to control phytopathogens, representing a key challenge in developing studies that translate basic into applied knowledge. During infection, X. citri subsp. citri secretes a transcriptional activator-like effector that enters the nucleus of plant cells, activating the expression of the canker susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1). In this study, we explored the use of antisense oligonucleotides (ASOs) with phosphorothioate modifications to transiently inhibit the gene expression of CsLOB1 in Citrus sinensis. We designed and validated three potential ASO sequences, which led to a significant reduction in disease symptoms compared to the control. The selected ASO3-CsLOB1 significantly decreased the expression level of CsLOB1 when delivered through two distinct delivery methods and the reduction of the symptoms ranged from approximately 15% to 83%. Notably, plants treated with ASO3 did not exhibit an increase in symptoms development over the evaluation period. This study highlights the efficacy of ASO technology, based on short oligonucleotide chemically modified sequences, as a promising tool for controlling phytopathogens without the need for genetic transformation or plant regeneration. Our results demonstrate the potential of ASOs as a biotechnological tool for the management of citrus canker disease.

ASOptimizer: Optimizing antisense oligonucleotides through deep learning for IDO1 gene regulation

Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered undruggable; however, manually designing optimal ASO sequences can be labor intensive and time consuming, which potentially limits their broader application. To address this challenge, we introduce a platform, the ASOptimizer, a deep-learning-based framework that efficiently designs ASOs at a low cost. This platform not only selects the most efficient mRNA target sites but also optimizes the chemical modifications for enhanced performance. Indoleamine 2,3-dioxygenase 1 (IDO1) promotes cancer survival by depleting tryptophan and producing kynurenine, leading to immunosuppression through the aryl-hydrocarbon receptor (Ahr) pathway within the tumor microenvironment. We used ASOptimizer to identify ASOs that target IDO1 mRNA as potential cancer therapeutics. Our methodology consists of two stages: sequence engineering and chemical engineering. During the sequence-engineering stage, we optimized and predicted ASO sequences that could target IDO1 mRNA efficiently. In the chemical-engineering stage, we further refined these ASOs to enhance their inhibitory activity while reducing their potential cytotoxicity. In conclusion, our research demonstrates the potential of ASOptimizer for identifying ASOs with improved efficacy and safety.

An Endosomal Escape Trojan Horse Platform to Improve Cytosolic Delivery of Nucleic Acids.

Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials. Here, we develop a pH-triggered spherical nucleic acid that provides smart antisense oligonucleotide (ASO) release upon endosomal acidification and selective membrane disruption, termed DNA EndosomaL Escape Vehicle Response (DELVR). We anchor i-Motif DNA to a nanoparticle (AuNP), where the complement strand contains both an ASO sequence and a functionalized endosomal escape peptide (EEP). By orienting the EEP toward the AuNP core, the EEP is inactive until it is released through acidification-induced i-Motif folding. In this study, we characterize a small library of i-Motif duplexes to develop a structure-switching nucleic acid sequence triggered by endosomal acidification. We evaluate antisense efficacy using HIF1a, a hypoxic indicator upregulated in many cancers, and demonstrate dose-dependent activity through RT-qPCR. We show that DELVR significantly improves ASO efficacy in vitro. Finally, we use fluorescence lifetime imaging and activity measurement to show that DELVR benefits synergistically from nuclease- and pH-driven release strategies with increased ASO endosomal escape efficiency. Overall, this study develops a modular platform that improves the cytosolic delivery of nucleic acid therapeutics and offers key insights for overcoming intracellular barriers.

Networking to Optimize Dmd exon 53 Skipping in the Brain of mdx52 Mouse Model.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the open reading frame and thus prevent production of functional dystrophin proteins. Recent advances in DMD treatment, notably exon skipping and AAV gene therapy, have achieved some success aimed at alleviating the symptoms related to progressive muscle damage. However, they do not address the brain comorbidities associated with DMD, which remains a critical aspect of the disease. The mdx52 mouse model recapitulates one of the most frequent genetic pathogenic variants associated with brain involvement in DMD. Deletion of exon 52 impedes expression of two brain dystrophins, Dp427 and Dp140, expressed from distinct promoters. Interestingly, this mutation is eligible for exon skipping strategies aimed at excluding exon 51 or 53 from dystrophin mRNA. We previously showed that exon 51 skipping can restore partial expression of internally deleted yet functional Dp427 in the brain following intracerebroventricular (ICV) injection of antisense oligonucleotides (ASO). This was associated with a partial improvement of anxiety traits, unconditioned fear response, and Pavlovian fear learning and memory in the mdx52 mouse model. In the present study, we investigated in the same mouse model the skipping of exon 53 in order to restore expression of both Dp427 and Dp140. However, in contrast to exon 51, we found that exon 53 skipping was particularly difficult in mdx52 mice and a combination of multiple ASOs had to be used simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2'MOE). Following ICV injection of a combination of ASO sequences, we measured up to 25% of exon 53 skipping in the hippocampus of treated mdx52 mice, but this did not elicit significant protein restoration. These findings indicate that skipping mouse dystrophin exon 53 is challenging. As such, it has not yet been possible to answer the pertinent question whether rescuing both Dp427 and Dp140 in the brain is imperative to more optimal treatment of neurological aspects of dystrophinopathy.

RNA-Based Strategies for Cancer Therapy: In Silico Design and Evaluation of ASOs for Targeted Exon Skipping.

Precision medicine in oncology has made significant progress in recent years by approving drugs that target specific genetic mutations. However, many cancer driver genes remain challenging to pharmacologically target ("undruggable"). To tackle this issue, RNA-based methods like antisense oligonucleotides (ASOs) that induce targeted exon skipping (ES) could provide a promising alternative. In this work, a comprehensive computational procedure is presented, focused on the development of ES-based cancer treatments. The procedure aims to produce specific protein variants, including inactive oncogenes and partially restored tumor suppressors. This novel computational procedure encompasses target-exon selection, in silico prediction of ES products, and identification of the best candidate ASOs for further experimental validation. The method was effectively employed on extensively mutated cancer genes, prioritized according to their suitability for ES-based interventions. Notable genes, such as NRAS and VHL, exhibited potential for this therapeutic approach, as specific target exons were identified and optimal ASO sequences were devised to induce their skipping. To the best of our knowledge, this is the first computational procedure that encompasses all necessary steps for designing ASO sequences tailored for targeted ES, contributing with a versatile and innovative approach to addressing the challenges posed by undruggable cancer driver genes and beyond.

In Vivo Imaging of [60]Fullerene-Based Molecular Spherical Nucleic Acids by Positron Emission Tomography.

18F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factor receptor 2 (HER2) mRNA antisense oligonucleotide sequence with a native phosphodiester and phosphorothioate backbone, were synthesized, site-specifically labeled with a positron emitting fluorine-18 and intravenously administrated via tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistribution of the MSNAs was monitored in vivo by positron emission tomography/computed tomography (PET/CT) imaging. MSNA with a native phosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediated degradation, whereas the corresponding phosphorothioate analogue (MSNA-PS) with improved enzymatic stability showed an interesting biodistribution profile in vivo. One hour after the injection, majority of the radioactivity was observed in spleen and liver but also in blood with an average tumor-to-muscle ratio of 2. The prolonged radioactivity in blood circulation may open possibilities to the targeted delivery of the MSNAs.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny.

Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

Systematic Analysis of Chemical Modifications of Phosphorothioate Antisense Oligonucleotides that Modulate Their Innate Immune Response.

While rare, some gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) can induce a noncanonical TLR9-dependent innate immune response. In this study, we performed systematic analyses of the roles of PS ASO backbone chemistry, 2' modifications, and sequence in PS ASO induced TLR9 signaling. We found that each of these factors can contribute to altering PS ASO induced TLR9 signaling, and in some cases the effects are quite dramatic. We also found that the positioning (5' vs. 3') of a particular backbone or 2' modification within a PS ASO can affect its TLR9 signaling. Interestingly, medicinal chemical strategies that decrease TLR9 signaling for one sequence can have opposing effects on another sequence. Our results demonstrate that TLR9 signaling is highly PS ASO sequence dependent, the mechanism of which remains unknown. Despite this, we determined that placement of two mesyl phosphoramidate linkages within the PS ASO gap is the most promising strategy to mitigate PS ASO dependent TLR9 activation to enhance the therapeutic index and, therefore, further streamline PS ASO drug development.

Cyclodextrin-Based Nanoparticles for Delivery of Antisense Oligonucleotides Targeting Huntingtin.

Huntington's disease (HD) is a progressive inherited neurodegenerative disease caused by a CAG repeat expansion in the huntingtin gene, which is translated into the pathologic mutant huntingtin (mHTT) protein. Despite the great potential of HTT lowering strategies and the numerous antisense oligonucleotides (ASOs) in pre- and clinical trials, sustained silencing of mHTT has not been achieved. As a strategy to improve ASO delivery, cyclodextrin-based nanoparticles (CDs) offer a promising approach. Here, three CDs with distinct chemical structures were designed and their efficacies were compared as potential platforms for the delivery of ASO targeting HTT. Results using striatal neurons and HD patient-derived fibroblasts indicate that modified γ-CDs exhibited the best uptake efficiency and successfully downregulated mHTT at protein and allele levels. The incorporation of the brain-targeting peptide RVG into the modified γ-CDs showed greater downregulation of mHTT protein and HD-causing allele SNP1 than untargeted ones in an in vitro blood-brain barrier model. Although the ASO sequence was designed as a nonallele-specific therapeutic approach, our strategy gives an additional benefit of some mHTT selectivity. Overall, this study demonstrated the CD platform's feasibility for delivering ASO-based therapeutics for HD treatment.

Correlations between preclinical BJAB assay ranking of antisense drugs and clinical trial adverse events.

This analysis sought to assess the clinical predictivity of an in vitro assay which utilized the human B-lymphoma BJAB cell line, for identification of antisense oligonucleotides (ASOs) with the potential to elicit innate immune activation in humans. Adverse events (AEs) from clinical trial data were analyzed based on prior clinical knowledge and network analysis of the clinical data to identify correlations with the BJAB assay. Clinically evaluated ASOs were ranked by the BJAB assay's mean log-fold increase in TNF expression levels. Flu-like reactions (FLRs) and injection site reactions (ISRs), were chosen as AEs of interest, along with those Medical Dictionary for Regulatory Activities preferred terms identified using AE network analysis. Fifteen different 2'-O-methoxyethyl (2'MOE) modified ASOs were ranked by the incidence of each AE group in the integrated safety data from 35 clinical trials. ISRs are considered to be local to the injection site, whereas FLRs are reflected by systemic constitutional symptoms. The correlations identified in this analysis of integrated clinical data provide evidence that the ASO sequences selected by the BJAB assay have a lower likelihood of causing systemic inflammatory AEs associated with FLRs, but not ISRs.

Antisense oligonucleotide induced pseudoexon skipping and restoration of functional protein for Fukuyama muscular dystrophy caused by a deep-intronic variant.

Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3'-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2'-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells, and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, ASO-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.

Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models

Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

Multi-pathway inducing ferroptosis by MnO2-based nanodrugs for targeted cancer therapy.

A multifunctional nanodrug (Tf-DHA-ASO-MnO2) based on manganese dioxide nanosheets was constructed by triple-dressing with transferrin, dihydroartemisinin, and antisense oligonucleotide sequences. Tf-DHA-ASO-MnO2 shows an effective targeted cancer therapy ability through the ferroptosis caused by the production of excessive lipid peroxides resulting from the combined effect of glutathione exhaustion, reactive oxygen species generation and down-regulation of glutathione peroxidase 4.

N-gene-complementary antisense-oligonucleotide directed molecular aggregation of dual-colour carbon dots, leading to efficient fluorometric sensing of SARS-COV-2 RNA.

The early stages of the COVID-19 pandemic punctuated the need for rapid, mass testing for early detection of viral infection. Carbon dots are easily synthesized, cost-effective fluorescent nanoparticles whose surface functionalities enable facile conjugation with biorecognition elements suitable for molecular detection of viral RNA. Herein, we report that a pair of complementary antisense oligonucleotide (ASO) sequences can lead to a highly specific molecular aggregation of dual colour carbon dots (CDs) in the presence of SARS-CoV-2 RNA. The nanoprobes used ASOs highly specific to the N-gene of SARS-COV-2. When the ASOs are conjugated to blue and yellow citric acid-derived CDs, the combination of the ASO-CD pairs facilitates aggregation-induced emission enhancement (AIEE) of the measured fluorescence after hybridization with SARS-CoV-2 RNA. We found the sensor capable of differentiating between MERS-CoV and SARS-CoV-2 samples and was found to have a limit of detection of 81 copies per μL. Additionally, we used dialysis to demonstrate that the change in emission upon aggregation is dependent on the compositional heterogeneity of the conjugated-carbon dot mixture.

Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52.

Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with >80% skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function.

Multiple antisense oligonucleotides targeted against monoacylglycerol acyltransferase 1 (Mogat1) improve glucose metabolism independently of Mogat1

ObjectiveMonoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of Mogat1 mRNA, which encodes MGAT1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high-fat diet (HFD)-fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway. MethodsMice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on an HFD, and glucose metabolism and insulin sensitivity were assessed after 16 weeks on diet. In some experiments, mice were treated with control scramble or Mogat1 ASOs in the presence or absence of IFNAR-1 neutralizing antibody. ResultsGenetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced obesity, insulin sensitivity, and glucose intolerance on an HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression in hepatocytes increased liver MGAT activity and TAG content in low-fat-fed mice but did not cause insulin resistance. Multiple Mogat1 ASO sequences improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off-target effect. Hepatic IFNAR-1 signaling was activated by multiple Mogat1 ASOs, but its blockade did not prevent the effects of either Mogat1 ASO on glucose homeostasis. ConclusionThese results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that multiple Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling.

Exosome-mediated delivery of antisense oligonucleotides targeting α-synuclein ameliorates the pathology in a mouse model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disease. Pathologically, PD is characterized by the formation of Lewy bodies (LBs) in the brain, which mainly comprises phosphorylated and aggregated α-synuclein (α-syn). The aberrant aggregation of α-syn is believed to play a key role in the pathogenesis of PD. While α-syn expression can be reduced by antisense oligonucleotides (ASOs), the challenge to deliver ASOs safely and effectively into the neurons remains unresolved. Here, we developed a safe and highly effective ASO delivery method by using exosomes. We first identified the ASO sequence that selectively reduced α-syn expression: ASO4. Exosome-mediated delivery of ASO4 (exo-ASO4) showed high cellular uptake and low toxicity in primary neuronal cultures. Exo-ASO4 also significantly attenuated α-syn aggregation induced by pre-formed α-syn fibrils in vitro. Exo-ASO4 intracerebroventricular injection into the brains of α-syn A53T mice, a transgenic model of PD, significantly decreased the expression of α-syn and attenuated its aggregation. Furthermore, exo-ASO4 ameliorated the degeneration of dopaminergic neurons in these mice. Finally, the α-syn A53T mice showed significantly improved locomotor functions after exo-ASO4 injection. Overall, this study demonstrates that exosome-mediated ASO4 delivery may be an effective treatment option for PD.

Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides.

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2′-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2′-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.

Albumin-Binding Fatty Acid-Modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics.

Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2′-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2′-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5′ region of exon 51. These results indicated that locked nucleic acid/2′-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.