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.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

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.

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.

Neurofilament Light Chain as a Biomarker of Hereditary Transthyretin-Mediated Amyloidosis.

OBJECTIVE: To identify changes in the proteome associated with onset and progression of hereditary transthyretin-mediated (hATTR) amyloidosis, also known as ATTRv amyloidosis, we performed an observational, case-controlled study that compared proteomes of patients with ATTRv amyloidosis and healthy controls. METHODS: Plasma levels of >1,000 proteins were measured in patients with ATTRv amyloidosis with polyneuropathy who received either placebo or patisiran in a Phase 3 study of patisiran (APOLLO), and in healthy controls. The effect of patisiran on the time profile of each protein was determined by linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) was further assessed with an orthogonal quantitative approach. RESULTS: Levels of 66 proteins were significantly changed with patisiran vs placebo, with NfL change most significant (p < 10-20). Analysis of changes in protein levels demonstrated that the proteome of patients treated with patisiran trended toward that of healthy controls at 18 months. Healthy controls' NfL levels were 4-fold lower than in patients with ATTRv amyloidosis with polyneuropathy (16.3 pg/mL vs 69.4 pg/mL, effect -53.1 pg/mL [95% confidence interval -60.5 to -45.9]). NfL levels at 18 months increased with placebo (99.5 pg/mL vs 63.2 pg/mL, effect 36.3 pg/mL [16.5-56.1]) and decreased with patisiran treatment (48.8 pg/mL vs 72.1 pg/mL, effect -23.3 pg/mL [-33.4 to -13.1]) from baseline. At 18 months, improvement in modified Neuropathy Impairment Score +7 score after patisiran treatment significantly correlated with reduced NfL (R = 0.43 [0.29-0.55]). CONCLUSIONS: Findings suggest that NfL may serve as a biomarker of nerve damage and polyneuropathy in ATTRv amyloidosis, enable earlier diagnosis of patients with ATTRv amyloidosis, and facilitate monitoring of disease progression. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that NfL levels may enable earlier diagnosis of polyneuropathy in patients with ATTRv amyloidosis and facilitate monitoring of disease progression.

Constraining Solar Wind Heating Processes by Kinetic Properties of Heavy Ions.

We analyze the heavy ion components (A>4 amu) in collisionally young solar wind plasma and show that there is a clear, stable dependence of temperature on mass, probably reflecting the conditions in the solar corona. We consider both linear and power law forms for the dependence and find that a simple linear fit of the form T_{i}/T_{p}=(1.35±.02)m_{i}/m_{p} describes the observations twice as well as the equivalent best fit power law of the form T_{i}/T_{p}=(m_{i}/m_{p})^{1.07±.01}. Most importantly we find that current model predictions based on turbulent transport and kinetic dissipation are in agreement with observed nonthermal heating in intermediate collisional age plasma for m/q<3.5, but are not in quantitative or qualitative agreement with the lowest collisional age results. These dependencies provide new constraints on the physics of ion heating in multispecies plasmas, along with predictions to be tested by the upcoming Solar Probe Plus and Solar Orbiter missions to the near-Sun environment.

GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-ß molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.

An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development.

MESSENGER observations of the spatial distribution of planetary ions near Mercury.

Global measurements by MESSENGER of the fluxes of heavy ions at Mercury, particularly sodium (Na(+)) and oxygen (O(+)), exhibit distinct maxima in the northern magnetic-cusp region, indicating that polar regions are important sources of Mercury's ionized exosphere, presumably through solar-wind sputtering near the poles. The observed fluxes of helium (He(+)) are more evenly distributed, indicating a more uniform source such as that expected from evaporation from a helium-saturated surface. In some regions near Mercury, especially the nightside equatorial region, the Na(+) pressure can be a substantial fraction of the proton pressure.