Bi-functional CpG-STAT3 decoy oligonucleotide triggers multilineage differentiation of acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.

Systemic Brain Delivery of Oligonucleotide Therapeutics Enhanced by Protein Corona-Assisted DNA Cubes.

The systemic delivery of oligonucleotide therapeutics to the brain is challenging but highly desirable for the treatment of brain diseases undruggable with traditional small-molecule drugs. In this study, a set of DNA nanostructures is prepared and screened them to develop a protein corona-assisted platform for the brain delivery of oligonucleotide therapeutics. The biodistribution analysis of intravenously injected DNA nanostructures reveals that a cube-shaped DNA nanostructure (D-Cb) can penetrate the brain-blood barrier (BBB) and reach the brain tissue. The brain distribution level of D-Cb is comparable to that of other previous nanoparticles conjugated with brain-targeting ligands. Proteomic analysis of the protein corona formed on D-Cb suggests that its brain distribution is driven by endothelial receptor-targeting ligands in the protein corona, which mediate transcytosis for crossing the BBB. D-Cb is subsequently used to deliver an antisense oligonucleotide (ASO) to treat glioblastoma multiforme (GBM) in mice. While free ASO is unable to reach the brain, ASO loaded onto D-Cb is delivered efficiently to the brain tumor region, where it downregulates the target gene and exerts an anti-tumor effect on GBM. D-Cb is expected to serve as a viable platform based on protein corona formation for systemic brain delivery of oligonucleotide therapeutics.

Intracellular Delivery of Antisense Oligonucleotides by Tri-Branched Cyclic Disulfide Units.

Therapeutic oligonucleotides, such as antisense DNA, show promise in treating previously untreatable diseases. However, their applications are still hindered by the poor membrane permeability of naked oligonucleotides. Therefore, it is necessary to develop efficient methods for intracellular oligonucleotide delivery. Previously, our group successfully developed disulfide-based Membrane Permeable Oligonucleotides (MPON), which achieved enhanced cellular uptake and gene silencing effects through an endocytosis-free uptake mechanism. Herein, we report a new molecular design for the next generation of MPON, called trimer MPON. The trimer MPON consists of a tri-branched backbone, three α-lipoic acid units, and a spacer linker between the oligonucleotides and tri-branched cyclic disulfide unit. We describe the design, synthesis, and functional evaluation of the trimer MPON, offering new insights into the molecular design for efficient oligonucleotide delivery.

Recent advances in methods for quantifying the cell penetration of macromolecules.

As the landscape of macromolecule therapeutics advances, drug developers are continuing to aim at intracellular targets. To activate, inhibit, or degrade these targets, the macromolecule must be delivered efficiently to intracellular compartments. Quite often, there is a discrepancy between binding affinity in biochemical assays and activity in cell-based assays. Identifying the bottleneck for cell-based activity requires robust assays that quantify total cellular uptake and/or cytosolic delivery. Recognizing this need, chemical biologists have designed a plethora of assays to make this measurement, each with distinct advantages and disadvantages. In this review, we describe the latest and most promising developments in the last 3 to 4 years.

Review of the Different Outcomes Produced by Genetic Knock Out of the Long Non-coding microRNA-host-gene MIR22HG versus Pharmacologic Antagonism of its Intragenic microRNA product miR-22-3p.

BACKGROUND: Publications reveal different outcomes achieved by genetically knocking out a long non-coding microRNA-host-gene (lncMIRHG) versus the administration of pharma-cologic antagomirs specifically targeting the guide strand of such intragenic microRNA. This suggests that lncMIRHGs may perform diverse functions unrelated to their role as intragenic miRNA precursors. OBJECTIVE: This review synthesizes in silico, in vitro, and in vivo findings from our lab and others to compare the effects of knocking out the long non-coding RNA MIR22HG, which hosts miR-22, versus administering pharmacological antagomirs targeting miR-22-3p. METHODS: In silico analyses at the gene, pathway, and network levels reveal both distinct and overlapping targets of hsa-miR-22-3p and its host gene, MIR22HG. While pharmacological an-tagomirs targeting miR-22-3p consistently improve various metabolic parameters in cell culture and animal models across multiple studies, genetic knockout of MIR22HG yields inconsistent results among different research groups. RESULTS: Additionally, MIR22HG functions as a circulating endogenous RNA (ceRNA) or "sponge" that simultaneously modulates multiple miRNA-mRNA interactions by competing for binding to several miRNAs. CONCLUSIONS: From a therapeutic viewpoint, genetic inactivation of a lncMIRHG and pharmaco-logic antagonism of the guide strand of its related intragenic miRNA produce different results. This should be expected as lncMIRHGs play dual roles, both as lncRNA and as a source for primary miRNA transcripts.

ASP210: a potent oligonucleotide-based inhibitor effective against TKI-resistant CML cells.

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.

Multiplexed In Vivo Screening Using Barcoded Aptamer Technology to Identify Oligonucleotide-Based Targeting Reagents.

Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for in vivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially in vivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and in vivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening in vivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.

Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

Preparing and Evaluating the Stability of Therapeutically Relevant Oligonucleotide Duplexes.

The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively. Key features • Adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other unmodified and modified oligonucleotides. • Does not necessitate any Biological Safety Level clearance and offers a rapid, cost-effective, and entirely in vitro procedure. • Allows researchers to evaluate multiple modification patterns that, when coupled with targeting activity, allow for selecting the best modification pattern prior to in vivo analysis.

Engineering cGAS-agonistic oligonucleotides as therapeutics for cancer immunotherapy.

Activating cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) holds great potential for cancer immunotherapy by eliciting type-I interferon (IFN-I) responses. Yet, current approaches to cGAS-STING activation rely on STING agonists, which suffer from difficult formulation, poor pharmacokinetics, and marginal clinical therapeutic efficacy. Here, we report nature-inspired oligonucleotide, Svg3, as a cGAS agonist for cGAS-STING activation in tumor combination immunotherapy. The hairpin-shaped Svg3 strongly binds to cGAS and enhances phase separation to form Svg3-cGAS liquid-like droplets. This results in cGAS-specific immunoactivation and robust IFN-I responses. Remarkably, Svg3 outperforms several state-of-the-art STING agonists in murine and human cells/tissues. Nanoparticle-delivered Svg3 reduces tumor immunosuppression and potentiates immune checkpoint blockade therapeutic efficacy of multiple syngeneic tumor models in wild-type mice, but in neither cGas-/- nor Sting-/- mice. Overall, these results demonstrate the great potential of Svg3 as a cGAS agonistic oligonucleotide for cancer combination immunotherapy.

Silencing Apoe with divalent-siRNAs improves amyloid burden and activates immune response pathways in Alzheimer's disease.

INTRODUCTION: The most significant genetic risk factor for late-onset Alzheimer's disease (AD) is APOE4, with evidence for gain- and loss-of-function mechanisms. A clinical need remains for therapeutically relevant tools that potently modulate APOE expression. METHODS: We optimized small interfering RNAs (di-siRNA, GalNAc) to potently silence brain or liver Apoe and evaluated the impact of each pool of Apoe on pathology. RESULTS: In adult 5xFAD mice, siRNAs targeting CNS Apoe efficiently silenced Apoe expression and reduced amyloid burden without affecting systemic cholesterol, confirming that potent silencing of brain Apoe is sufficient to slow disease progression. Mechanistically, silencing Apoe reduced APOE-rich amyloid cores and activated immune system responses. DISCUSSION: These results establish siRNA-based modulation of Apoe as a viable therapeutic approach, highlight immune activation as a key pathway affected by Apoe modulation, and provide the technology to further evaluate the impact of APOE silencing on neurodegeneration.

Dendritic amphiphilic siRNA: Selective albumin binding, in vivo efficacy, and low toxicity.

Although an increasing number of small interfering RNA (siRNA) therapies are reaching the market, the challenge of efficient extra-hepatic delivery continues to limit their full therapeutic potential. Drug delivery vehicles and hydrophobic conjugates are being used to overcome the delivery bottleneck. Previously, we reported a novel dendritic conjugate that can be appended efficiently to oligonucleotides, allowing them to bind albumin with nanomolar affinity. Here, we explore the ability of this novel albumin-binding conjugate to improve the delivery of siRNA in vivo. We demonstrate that the conjugate binds albumin exclusively in circulation and extravasates to various organs, enabling effective gene silencing. Notably, we show that the conjugate achieves a balance between hydrophobicity and safety, as it significantly reduces the side effects associated with siRNA interactions with blood components, which are commonly observed in some hydrophobically conjugated siRNAs. In addition, it reduces siRNA monocyte uptake, which may lead to cytokine/inflammatory responses. This work showcases the potential of using this dendritic conjugate as a selective albumin binding handle for the effective and safe delivery of nucleic acid therapeutics. We envision that these properties may pave the way for new opportunities to overcome delivery hurdles of oligonucleotides in future applications.

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.

Asymmetric trichotomous partitioning overcomes dataset limitations in building machine learning models for predicting siRNA efficacy.

Chemically modified small interfering RNAs (siRNAs) are promising therapeutics guiding sequence-specific silencing of disease genes. Identifying chemically modified siRNA sequences that effectively silence target genes remains challenging. Such determinations necessitate computational algorithms. Machine learning is a powerful predictive approach for tackling biological problems but typically requires datasets significantly larger than most available siRNA datasets. Here, we describe a framework applying machine learning to a small dataset (356 modified sequences) for siRNA efficacy prediction. To overcome noise and biological limitations in siRNA datasets, we apply a trichotomous, two-threshold, partitioning approach, producing several combinations of classification threshold pairs. We then test the effects of different thresholds on random forest machine learning model performance using a novel evaluation metric accounting for class imbalances. We identify thresholds yielding a model with high predictive power, outperforming a linear model generated from the same data, that was predictive upon experimental evaluation. Using a novel model feature extraction method, we observe target site base importances and base preferences consistent with our current understanding of the siRNA-mediated silencing mechanism, with the random forest providing higher resolution than the linear model. This framework applies to any classification challenge involving small biological datasets, providing an opportunity to develop high-performing design algorithms for oligonucleotide therapies.

Engineering cGAS-agonistic oligonucleotides as therapeutics and vaccine adjuvants for cancer immunotherapy.

Current cancer immunotherapy (e.g., immune checkpoint blockade (ICB)) has only benefited a small subset of patients. Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) activation holds the potential to improve cancer immunotherapy by eliciting type-I interferon (IFN-I) responses in cancer cells and myeloid cells. Yet, current approaches to this end, mostly by targeting STING, have marginal clinical therapeutic efficacy. Here, we report a cGAS-specific agonistic oligonucleotide, Svg3, as a novel approach to cGAS-STING activation for versatile cancer immunotherapy. Featured with a hairpin structure with consecutive guanosines flanking the stem, Svg3 binds to cGAS and enhances cGAS-Svg3 phase separation to form liquid-like droplets. This results in cGAS activation by Svg3 for robust and dose-dependent IFN-I responses, which outperforms several state-of-the-art STING agonists in murine and human immune cells, and human tumor tissues. Nanocarriers efficiently delivers Svg3 to tissues, cells, and cytosol where cGAS is located. Svg3 reduces tumor immunosuppression and potentiates ICB therapeutic efficacy of multiple syngeneic tumors, in wildtype but neither cGas-/- nor goldenticket Sting-/- mice. Further, as an immunostimulant adjuvant, Svg3 enhances the immunogenicity of peptide antigens to elicit potent T cell responses for robust ICB combination immunotherapy of tumors. Overall, cGAS-agonistic Svg3 is promising for versatile cancer combination immunotherapy.

Deep intronic founder mutations identified in the ERCC4/XPF gene are potential therapeutic targets for a high-frequency form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.

Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

The structure and function of YTHDF epitranscriptomic m6A readers.

Specific RNA sequences modified by a methylated adenosine, N6-methyladenosine (m6A), contribute to the post-transcriptional regulation of gene expression. The quantity of m6A in RNA is orchestrated by enzymes that write and erase it, while its effects are mediated by proteins that bind to read this modification. Dysfunction of this post-transcriptional regulatory process has been linked to human disease. Although the initial focus has been on pharmacological targeting of the writer and eraser enzymes, interest in the reader proteins has been challenged by a lack of clear understanding of their functional roles and molecular mechanisms of action. Readers of m6A-modified RNA (m6A-RNA) - the YTH (YT521-B homology) domain-containing protein family paralogs 1-3 (YTHDF1-3, referred to here as DF1-DF3) - are emerging as therapeutic targets as their links to pathological processes such as cancer and inflammation and their roles in regulating m6A-RNA fate become clear. We provide an updated understanding of the modes of action of DF1-DF3 and review their structures to unlock insights into drug design approaches for DF paralog-selective inhibition.

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.

Acyclic (S)-glycol nucleic acid (S-GNA) modification of siRNAs improves the safety of RNAi therapeutics while maintaining potency.

Glycol nucleic acid (GNA) is an acyclic nucleic acid analog connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base-pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.