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.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

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.

Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing.

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using 'click' chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5'-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

Spinning the globe from west to east: A mixed-method study to examine the impact of pharmacists on immunization advocacy and delivery in Asia Pacific.

BACKGROUND: Global effort is key to prevent and control infections effectively. Whereas pharmacists' involvement in the immunization neighborhood, collaborative effort to meet immunization needs of the public, has reduced burden of vaccine-preventable diseases in Western countries, the impact of pharmacist-involved vaccination advocacy and delivery in Asia Pacific remains unclear. OBJECTIVE: To examine the evidence for impact of pharmacists on immunization advocacy and delivery in Asia Pacific and to assess Singaporean pharmacists' experience in becoming immunizing pharmacists through the American Pharmacists Association's Pharmacy-based Immunization certificate training program. METHODS: A mixed-method study consisted of systematic review and survey was conducted. The systematic review was conducted by searching PubMed and Cumulative Index to Nursing and Allied Health databases from inception to March 2021. Randomized controlled trials and observational studies related to pharmacist-involved immunization advocacy and training conducted in Asia Pacific were included. Eligible studies were appraised for quality using CONsolidated Standards of Reporting Trials (CONSORT) and STrengthening the Reporting of OBservational checklists. In addition, a 20-item Likert-scale survey evaluated the pharmacists' experience in becoming immunizing pharmacists in Singapore. Frequencies of each response to the survey items were analyzed. RESULTS: Six studies were eligible for inclusion; 2 assessing impact of pharmacist as immunizer, 3 reporting outcomes of pharmacist-involved vaccine advocacy, and 1 evaluating the structure of pharmacist immunization training. Although study quality was minimal, all studies echoed positive findings as a result of pharmacist-provided immunization advocacy and delivery. Of the 23 pharmacists completing the survey, 91.3% strongly agreed that quality training was important to become immunizing pharmacists. CONCLUSION: Although limited in number, studies conducted in Asia Pacific reported increased vaccination rates when involving pharmacists. Coupled with the enthusiasm to be trained in immunization advocacy and delivery as elucidated by the Singaporean pharmacists, pharmacists in Asia Pacific should also be advocated for their role in the immunization neighborhood as part of the global effort against vaccine-preventable infectious diseases.

The Role of Polyphenols in Human Health and Food Systems: A Mini-Review.

This narrative mini- review summarizes current knowledge of the role of polyphenols in health outcomes-and non-communicable diseases specifically-and discusses the implications of this evidence for public health, and for future directions for public health practice, policy, and research. The publications cited originate mainly from animal models and feeding experiments, as well as human cohort and case-control studies. Hypothesized protective effects of polyphenols in acute and chronic diseases, including obesity, neurodegenerative diseases, type 2 diabetes, and cardiovascular diseases, are evaluated. Potential harmful effects of some polyphenols are also considered, counterbalanced with the limited evidence of harm in the research literature. Recent international governmental regulations are discussed, as the safety and health claims of only a few specific polyphenolic compounds have been officially sanctioned. The implications of food processing on the bioavailability of polyphenols are also assessed, in addition to the health claims and marketing of polyphenols as a functional food. Finally, this mini-review asserts the need for increased regulation and guidelines for polyphenol consumption and supplementation in order to ensure consumers remain safe and informed about polyphenols.