Similar in structure to both glycol nucleic acid (GNA) and peptide nucleic acid (PNA), serinol nucleic acid (SNA) combines the acyclic flexibility of GNA with the amide bond found in PNA and can form stable heteroduplexes with DNA and RNA. While the thermal and metabolic stability of SNA and SNA-modified heteroduplexes has been previously reported, we here describe the application of SNA in RNA interference (RNAi) using GalNAc-conjugated small interfering RNAs (siRNAs). Single incorporations were evaluated at each position across both the guide and passenger strands and subsequently evaluated at key positions in mice. Additionally, the off-target potential of siRNAs containing SNA was assessed. We demonstrate the position-dependent tolerance of SNA inside both the guide and passenger strands, and more specifically, high tolerance of SNA incorporation in the seed region of the guide strand, preserving on-target activity while mitigating microRNA-like off-target effects. Furthermore, the crystal structure of an RNA dodecamer containing a single SNA nucleotide was obtained and the resulting SNA structure was used to explain the activity of SNA containing siRNAs. Thus, SNA constitutes another viable modification in the siRNA toolbox for the development of potent and specific RNAi therapeutics.

Unlocked nucleic acid (UNA) is a nucleic acid analog that has an acyclic ribose ring lacking the bond between C2' and C3' atoms. The base-pairing properties of UNA have been studied, and it has been used as a scaffold for conjugation, but the chemical space around UNA and its potential applications in the context of short interfering RNAs (siRNAs), which mediate RNA interference, have not been thoroughly explored. In this study, we report syntheses of methylated and methoxylated UNAs and their incorporation into siRNAs. siRNAs with 5'-(R)-methyl-UNA and with 5'-(S)-methyl-UNA in the seed region had comparable potencies but reduced off-target effects compared to siRNA modified with UNA. In mice, siRNAs with modified UNAs were of comparable potency to an siRNA of the same sequence and chemistry lacking UNA. Modeling studies indicated that the flexibilities of UNA and the modified UNAs facilitate kinking of the antisense strand when incorporated at position 7. These findings highlight the potential of modified UNA for advancing therapeutics that act through the RNA interference pathway.

The conjugation of the sense strands of small interfering RNA (siRNA) to tri-N-acetylgalactosamine (GalNAc), the ligand for a hepatocyte-specific receptor, enables the delivery of multiple clinically approved therapeutic agents that act through the RNA interference pathway. Here, we report the systematic evaluation of siRNAs with the 3 ' termini of antisense strands conjugated to GalNAc for the first time. These designs retained the same receptor affinity, in vitro and in vivo activities, as well as the same level of loading into the RNA-induced silencing complex as siRNAs with a GalNAc-conjugated sense strand. A siRNA with a GalNAc-conjugated antisense strand of 22 nucleotides had better activity than a siRNA with a 23-nucleotide antisense strand. Computational modeling of a complex of a GalNAc-conjugated antisense strand with the PAZ domain of Ago2 rationalizes the importance of the interaction of phosphate at the 3 ' terminus with the PAZ domain to explain the observed activity of these siRNAs.

Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested "Repair Drive", a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytes in vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.

The hyperphosphorylation, mislocalization, and aggregation of the microtubule associated protein Tau (MAPT) is a driving force in tauopathies, a group of progressive, neurodegenerative disorders. These pathogenic intracellular aggregates, known as neurofibrillary tangles (NFTs), are a hallmark in several diseases such as frontotemporal dementia, progressive supranuclear palsy, and Alzheimer’s Disease. While anti-Tau immunotherapies emphasize the clearance of extracellular Tau aggregates, they do not address the intracellular accumulation of NFTs. Here, using our clinically validated C16-siRNA CNS delivery platform, we have identified potent molecules targeting the MAPT gene. We have identified potent molecules that durably reduce MAPT mRNA and Tau protein in both in vitro and in vivo model systems. Furthermore, in a non-human primate study, we demonstrated robust, sustained reduction of MAPT transcript and tau protein in disease relevant brain tissue that was well-tolerated through 16-weeks. Together, these results suggest that Tau-lowering via C16-siRNAi may provide a novel strategy to reduce, and potentially reverse, accumulation of intracellular and extracelluar Tau aggregates. This approach has advanced into in IND-enabling development.

Chemical modifications are applied to small interfering RNAs (siRNAs) to improve their metabolic stability, specificity, and duration of pharmacodynamic effects. Despite tremendous progress made, identifying chemically modified siRNAs with drug-like properties requires empirical screening due to an intricate interdependence of siRNA sequence and chemistry, i.e., the nature and position of chemical modifications within the siRNA duplex. To improve our ability to design fully modified, potent siRNAs, we combined experimental measurements of thermodynamic stability and biological activity in vitro with extensive molecular modeling in silico of the structural, dynamic, and energetic properties of parent (unmodified) siRNA duplex sequences compared with their chemically modified variants. A pattern of modifications at specific positions were identified, where the combination of sequence and chemical modifications play an outsized role in the observed biological activity. Molecular modeling revealed low stabilization energies and increased sugar stereochemical flexibility for 20-F modified position g2 and less so for g6 in the guide strand seed region. Machine learning confirmed that these properties correlate with higher observed biological activity. These results provide molecular-level insights into the effects of chemical modifications on the intrinsic activity of siRNAs, which can be used in the rational design of chemically modified siRNAs with uncompromised potency.

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral RNA. We identified potent lipophilic small interfering RNA (siRNA) conjugates targeting highly conserved regions of SARS-CoV-2 outside of the spike-encoding region capable of achieving ≥3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single siRNA approach. Viral resistance to single siRNA treatment occurred due to emergence of point mutations at critical positions required for siRNA-mediated target binding and cleavage, which led to a loss of siRNA efficacy. With a two-siRNA combination, emergence of mutations within the siRNA binding site was abolished. When delivered intranasally, two-siRNA combination protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach with high resistance barrier to counteract SARS-CoV-2 emergent variants and complement vaccination. Most importantly, given that the siRNAs can be rapidly developed from a new pathogen sequence, this strategy has implications as a new type of preventive medicine that may protect against future coronavirus pandemics.

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5 '-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His(6)-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2 '-3 ' linked alpha-L-threofuranosyl thymidine-3 '-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (K-d) of MID-nucleotide interactions for nucleoside 5 '-monophosphates and nucleoside 3 ',5 '-bisphosphates. (c) 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

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.

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.

The hyperphosphorylation and subsequent aggregation of the microtubule-associated protein Tau (encoded by the MAPT gene) is one of the major histopathological hallmarks of numerous tauopathies, including Alzheimer’s disease and progressive supranuclear palsy. The intracellular accumulation of aggregated Tau is associated with neuronal loss and disease progression in tauopathies. There is a high-unmet need to identify strategies to target the intracellular accumulation of Tau aggregates with the aim of slowing tauopathy progression. To model this, we used mice carrying multiple copies of a transgene with a prion promoter driving human MAPT with human tauopathy-inducing mutation P301S. P301S-Tau transgenic mice progressively accumulate neuroglial Tau aggregates associated with axonal damage, gliosis, body weight loss, and ambulatory dysfunction. P301S mice treated with a single intracerebroventricular injection of Tau siRNA at eight months of age, had a sustainable decrease in MAPT mRNA and soluble Tau protein for three months following siRNA treatment (reduced ∼83% and ∼59%, respectively). siRNA treatment also prevented the progressive decrease in body weight and abrogated axonal damage, evidenced by decreased neurofilament light (NFL) chain in plasma and cerebrospinal fluid of eleven-month-old mice. Importantly, immunohistochemical analysis of misfolded Tau in vivo and HEK293-4RD-CFP/YFP Tau aggregate biosensor assay testing in vitro revealed a ∼97% decrease of Tau aggregates in the eleven-month-old mice, suggesting there exists a yet-to-be identified mechanism of Tau fibril clearance. Together, these results suggest an RNAi therapeutics approach targeting of MAPT mRNA may be sufficient to improve multiple parameters of tauopathy disease progression and a provide a compelling rationale for further development of Tau-lowering strategies.

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral genome. We screened lipophilic small-interfering RNA (siRNA) conjugates targeting highly conserved regions of the SARS-CoV-2 genome and identified leads targeting outside of the spike-encoding region capable of achieving ≥3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single-siRNA approach. A two-siRNA combination delivered intranasally protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach to limit SARS-CoV-2 infection that may help combat emergent variants, complement existing interventions, or protect populations where vaccines are less effective. Most importantly, this strategy has implications for developing medicines that may be valuable in protecting against future coronavirus pandemics.

Abstract 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.

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.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

RNA interference (RNAi) therapeutics are a new class of medicines that can address unmet medical needs by silencing disease-causing gene transcripts. While delivery of short interfering RNAs (siRNAs) to hepatocytes has yielded multiple drug approvals, novel delivery solutions are needed to expand the reach of RNAi therapeutics. Here we report that conjugation of 2'- O -hexadecyl (C16) to siRNAs enables efficient silencing in the central nervous system (CNS), eye, and lung of multiple nonclinical species with broad cell type specificity. Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route. Similarly, intravitreal and intranasal administration of C16-siRNAs resulted in potent and sustained knockdown in the eye and lung, respectively. Efficient delivery facilitated through C16 conjugation to optimized siRNA designs has enabled candidate selection for investigational human clinical trials assessing therapeutic silencing beyond the liver with infrequent (e.g. bi-annual) dosing.

Abstract 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.

Toward the goal of evaluation of carbocyclic ribonucleoside-containing oligonucleotide therapeutics, we developed convenient, scalable syntheses of all four carbocyclic ribonucleotide phosphoramidites and the uridine solid-support building block. Crystallographic analysis confirmed configuration and stereochemistry of these building blocks. Duplexes with carbocyclic RNA (car-RNA) modifications in one strand were less thermodynamically stable than duplexes with unmodified RNA. However, circular dichroism spectroscopy indicated that global conformations of the duplexes containing car-RNAs were similar to those in the unmodified duplexes.