From bench to bedside: Improving the clinical safety of GalNAc-siRNA conjugates using seed-pairing destabilization.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-ß-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc-siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5'-exonuclease.

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification.

Weigh-in-Motion System Based on an Improved Kalman and LSTM-Attention Algorithm.

A weigh-in-motion (WIM) system continuously and automatically detects an object's weight during transmission. The WIM system is used widely in logistics and industry due to increasing labor and time costs. However, the accuracy and stability of WIM system measurements could be affected by shock and vibration under high speed and heavy load. A novel six degrees-of-freedom (DOF), mass-spring damping-based Kalman filter with time scale (KFTS) algorithm was proposed to filter noise due to the multiple-input noise and its frequency that is highly coupled with the basic sensor signal. Additionally, an attention-based long short-term memory (LSTM) model was built to predict the object's mass by using multiple time-series sensor signals. The results showed that the model has superior performance compared to support vector machine (SVM), fully connected network (FCN) and extreme gradient boosting (XGBoost) models. Experiments showed this improved deep learning model can provide remarkable accuracy under different loads, speed and working situations, which can be applied to the high-precision logistics industry.

Professional quality of life in nurses on the frontline against COVID-19.

AIMS: This study aimed to investigate professional quality of life (ProQOL) in nurses who were fighting against COVID-19 in Wuhan and its related factors. BACKGROUND: COVID-19 epidemic is a major threat to public health. Frontline nurses have engaged in infection prevention and control, isolation, containment and public health. However, available data on ProQOL in these nurses are limited. METHODS: From 15 to 21 March 2020, the Chinese version of ProQOL was utilized to survey a total of 102 nurses through an electronic questionnaire. The stepwise regression analysis was performed to determine which factors (e.g. demographic and work-related factors) were related to ProQOL. RESULTS: The scores of compassion satisfaction (CS), burnout (BO) and secondary traumatic stress (STS) were 38.09 ± 5.22, 21.77 ± 4.92 and 20.75 ± 6.27, respectively. The STS and CS scores were higher than the critical value. None of the nurses reported a low level of CS or a high level of BO and STS. Nurses' ProQOL was related to working hours, workload, job satisfaction and salary satisfaction. CONCLUSIONS: Nurses who were fighting against COVID-19 had better CS and BO, whereas STS was relatively worse. Nurses who worked for long hours had more severe STS. BO of nurses with heavy workload and dissatisfaction with their salary was more severe. Nurses who were unsatisfied with their job had poor CS. IMPLICATIONS FOR NURSING MANAGEMENT: It is believed that these results may help nurse managers to improve ProQOL of nurses who were fighting against COVID-19 by minimizing working hours, reducing workload and improving job satisfaction and rewards.

Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Rational optimization of siRNA to ensure strand bias in the interaction with the RNA-induced silencing complex.

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA.

Heat Stability and Icing Delay on Superhydrophobic Coatings in Facile One Step.

Superhydrophobic coatings are limited to poor durability and a tedious preparation process. In this work, an efficient, eco-friendly, and cost-effective sol-gel method is developed for preparing superhydrophobic surfaces using an all-in-one suspension composed of methyltrimethoxysilane (MTMS), nano silicon dioxide (SiO2) particles, and micron zinc oxide (ZnO) particles. Superhydrophobic coatings with a contact angle (CA) up to 153.9° and a sliding angle (SA) of about 3.0° are prepared on Q235 steel substrates using MTMS 5 mL, 0.8 g of nano SiO2, and 0.2 g of micron ZnO. The morphology of the superhydrophobic coating is characterized by scanning electron microscopy (SEM), and the surface is covered with a micro- and nano-scaled hierarchical rough structure. A series of tests are conducted, including long-term stability tests and thermostability tests. The CAs are all above 150°, and the SAs are below 6.3°, indicating the excellent static stability of the prepared superhydrophobic coatings. Moreover, the CA of the superhydrophobic coating remains above 152° after 120 h of UV exposure, and the time for a water droplet to freeze on the surface of the superhydrophobic coating is 18 times of the bare Q235 steel, indicating that the superhydrophobic coating exhibits good resistance to UV radiation and icing-delay properties.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

P2Y6 receptors require an intact cysteinyl leukotriene synthetic and signaling system to induce survival and activation of mast cells.

Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors and activate mast cells in vitro. We previously demonstrated that cys-LTs cross-desensitized IL-4-primed primary human mast cells (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. Leukotriene (LT) D4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of ERK and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase, implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of MIP-1beta, a response blocked by short hairpin RNA-mediated knockdown of CysLT1R or P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are coexpressed on many cell types of innate immunity, reciprocally amplify one another's function in mast cells through endogenous ligands.

Association of Peripheral Blood Levels of Cytokines With Autism Spectrum Disorder: A Meta-Analysis.

Background: Although increasing evidence suggests an association between alterations in peripheral cytokines and autism spectrum disorder (ASD), a consensus is lacking. To determine whether abnormal cytokine profiles in peripheral blood were associated with ASD, we performed this systemic review and meta-analysis. Methods: A systematic literature search was conducted through the Embase, PubMed, Web of Knowledge, PsycINFO, and Cochrane databases up to 4 June 2020. Clinical studies exploring the aberration of peripheral cytokines of autistic patients and controls were included in our meta-analysis. We pooled extracted data using fixed- or random-effects models based on heterogeneity tests with Comprehensive Meta-analysis software. We converted standardized mean differences to Hedges' g statistic to obtain the effect sizes adjusted for sample size. Subgroup analyses, sensitivity analyses, meta-regression, and publication bias tests were also carried out. Results: Sixty-one articles (326 studies) were included to assess the association between 76 cytokines and ASD. We conducted our meta-analysis based on 37 cytokines with 289 studies. Since there were fewer than three studies on any of the other 39 cytokines, we only provided basic information for them. The levels of peripheral IL-6, IL-1ß, IL-12p70, macrophage migration inhibitory factor (MIF), eotaxin-1, monocyte chemotactic protein-1 (MCP-1), IL-8, IL-7, IL-2, IL-12, tumor necrosis factor-α (TNF-α), IL-17, and IL-4 were defined as abnormal cytokines in the peripheral blood of ASD patients compared with controls. The other 24 cytokines did not obviously change in ASD patients compared with the controls. Conclusions: The findings of our meta-analysis strengthen the evidence for an abnormal cytokine profile in ASD. These abnormal cytokines may be potential biomarkers for the diagnosis and treatment of ASD in the future.

Management and experience of postural placement in postoperative mechanical ventilation of newborns.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a neonatal condition that mainly occurs when the abdominal organs herniate into the thorax, obstructing the development of the lungs. Postoperative neonatal breathing disorder is one of the main causes of neonatal death. This study summarizes the postoperative nursing status of 30 cases of neonatal CDH in our hospital, and explores the effect of body position in mechanical ventilation care following CDH surgery. METHODS: A total of 30 CHD children admitted in our hospital between June, 2018 and October, 2019 were included. The neonates were divided into preterm infant group (n=15) and full-term infant group (n=15). Each child was immediately transferred to the newborn intensive care unit (NICU) ward after surgery and received the hospital's NCIU special care. Besides routine nursing, each child was placed in a randomly selected primary position (the prone position or supine position). After 30 min, their oxygenation indexes were measured, and then their position (prone position or supine position) was changed. After 30 min, the neonates' oxygenation indicators were measured again. A hospital-made satisfaction questionnaire was used to evaluate the parents' satisfaction with nursing care. RESULTS: Two children died of respiratory failure (one in the preterm group and one in the full-term group), and the rest were in a stable condition after surgery. There was no significant difference between the rates of parent satisfaction in the two groups (preterm infant group: 96.67% verse full-term group: 93.33%, P>0.05). In both groups, the partial pressure of oxygen in arterial blood (PaO2), partial pressure of oxygen in arterial blood/fraction of inspiration O2 (PaO2/FiO2), and partial pressure of oxygen in arterial blood/partial pressure of oxygen in the alveolar gas (PaO2/PAO2) in prone position were significantly higher than those in supine position (P<0.05); the alveolar-arterial oxygen difference (A-aDO2) was significantly lower than that in the supine position (0.05). CONCLUSIONS: The prone position can improve the oxygenation index of children after surgery, and improve their respiratory system function. This method is suitable for newborn postoperative NICU care.

[Spatial Distribution, Spectral Characteristics, and Sources Analysis of Dissolved Organic Matter from Baiyangdian Lake in Xiong'an New District During the Winter Freezing Period].

Based on excitation emission matrix spectroscopy (EEMs) technology combined with parallel factor analysis (PARAFAC) and ultraviolet-visible (UV-vis) spectra, we analyze the spatial distribution, spectral characteristics, and sources of dissolved organic matter (DOM) in Baiyangdian Lake, China, during a winter freezing period. Results showed that the UV-vis absorption spectrum of DOM had no obvious characteristic peak, and that the variation coefficient of absorption exhibited a significant difference (P<0.05) among different districts of Baiyangdian Lake, but that there was no significant difference between the surface and bottom waters. The changes of E3/E4, E2/E3, and SR showed that DOM had low humic and autochthonous characteristics. Two protein-like substances (C1 and C2) and one humic-like substance (C3) were identified by PARAFAC, with a significant correlation (P<0.001) being found between C1 and C3, and C2 and C3. The total DOM fluorescence intensity and the fluorescence intensity of each component exhibited significant differences (P<0.01) in the distribution among the different districts of Baiyangdian Lake, with the maximum value being associated with a sample from the Tanghe River and the minimum value being associated with a sample from Shaochedian. Moreover, the sum of C1+C2 accounted for the major proportion of DOM. DOM exhibited a strong autochthonous characteristic based on the values of BIX, FI, and HIX. Principle component analysis (PCA) and Adonis analysis showed that the spectral characteristics of DOM exhibited a significant difference (P<0.05) among the different districts. C1, C2, and C3 were significantly correlated (P<0.001) with DOM indices (HIX, BIX, Fn280, and Fn355) and water quality parameters[total nitrogen (TN), permanganate index, and total dissolved phosphorus (TDP)] based on multiple linear regression. Our results contribute to previous investigations and provide findings that can assist in the future management and control of organic carbon pollution sources to Baiyangdian Lake.

Reaction kinetics of phenols and p-nitrophenols in flowing aerated aqueous solutions generated by a discharge plasma jet.

In this paper, we propose a method for removing phenols and p-nitrophenols (PNPs) from flowing aqueous solutions generated by atmospheric pressure plasma jets (APPJs). For analyzing the removal characteristics, multiple techniques were used, including flow speed analysis of the aerated solution, optical emission spectroscopy (OES), and liquid chromatography. In addition, the reaction kinetics of diffusion and activation control processes were evaluated using aerated fluid speed variation and the corresponding activation energy. From these results, the relative intensities of hydroxyl radicals produced by an APPJ in water were found to be stronger than those in air and to decrease with increasing flow speed. Furthermore, the reaction kinetics were found to be diffusion-controlled when the solution flow speed was low and activation-controlled under high solution flow speed. It was also found that the degradation efficiency was enhanced with increasing flow speed, which increased the discharge voltage and temperature of the solution and changed the initial pH value when TiO2/UV catalysis was used. From the complex relationship between the reactive species, fluid diffusion, and discharge parameters in wastewater described herein, it is anticipated that these findings will facilitate new approaches to both the design and optimization of discharge reactors intended for wastewater treatment.

Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity.

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.

Impact of Oligonucleotide Structure, Chemistry, and Delivery Method on In Vitro Cytotoxicity.

Single-stranded (ss) 2'-fluoro (2'-F)-modified oligonucleotides (ONs) with a full phosphorothioate (PS) backbone have been reported to be cytotoxic and cause DNA double-strand breaks (DSBs) when transfected into HeLa cells. However, the molecular determinants of these effects have not been fully explored. In this study, we investigated the impact of ON structure, chemistry, delivery method, and cell type on in vitro cytotoxicity and DSBs. We found that ss PS-ONs were more cytotoxic than double-stranded (ds) PS-ONs, irrespective of the 2'-ribose chemistry, inclusive of the 2'-F modification. Cytotoxicity of ss ONs was most affected by the total PS content, with an additional contribution of 2'-F substitutions in HeLa, but not HepG2, cells. The relatively mild cytotoxicity of ds ONs was most impacted by long contiguous PS stretches combined with 2'-F substitutions. None of the tested ds 2'-F-modified PS-ONs caused DSBs, while the previously reported DSBs caused by ss 2'-F-modified PS-ONs were PS dependent. HeLa cells were more sensitive to ON-mediated toxicity when transfected with Lipofectamine 2000 versus Lipofectamine RNAiMax. Importantly, asialoglycoprotein receptor-mediated uptake of N-acetylgalactosamine-conjugated ss or ds PS-ONs, even those with long PS stretches and high 2'-F content, was neither cytotoxic nor caused DSBs at transfection-equivalent exposures. These results suggest that in vitro cytotoxicity and DSBs associated with ONs are delivery method dependent and primarily determined by single-stranded nature and PS content of ONs.

Exposure to siRNA-GalNAc Conjugates in Systems of the Standard Test Battery for Genotoxicity.

Registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-O-methyl ribose modifications of multiple nucleotides in the ICH battery of assays, all of which have uniformly yielded negative results. To verify these negative genotoxicity results, in this study we confirm test article exposure using toolkit small interfering RNAs (siRNAs) representative of those in the clinic. In the Ames test, the highest uptake of the siRNA-GalNAc conjugates occurred at 1 h postdose in all bacterial strains independent of siRNA sequence or chemistry (up to ∼14,000 siRNA molecules per cell), followed by metabolic degradation of the parent siRNA at 6, 24, and 48 h postdose. siRNA-GalNAc conjugates were internalized by bacteria as assessed by protection from the addition of nucleases to the culture media following uptake and by the requirement of cell lysis for detection of the siRNA. In the in vitro chromosome aberration assay, uptake was observed in Chinese hamster ovary cells (up to ∼5,500 siRNA molecules per cell at 21 h postdose) and in CD3+ human peripheral blood lymphocytes (up to ∼500 siRNA molecules per cell at 21 h postdose). In the in vivo micronucleus assay in rat bone marrow, exposure to parent siRNA was 100-350 µg of antisense strand per gram of protein at 24 and 48 h postlimit dose of 2 g/kg. Loss of terminal nucleotides was detected in bone marrow by mass spectrometry, indicating exposure to monomer metabolites as well. Negative genotoxicity results were also confirmed in an in vitro double-strand DNA break assay in HeLa and HepG2 cells where exposure was maximized using transfection reagents. Thus negative genotoxicity assay results for siRNA-GalNAc conjugates were valid and not the result of poor or no intracellular exposure.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.