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.

5'-Morpholino modification of the sense strand of an siRNA makes it a more effective passenger.

The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

Identification of Filamin-A and -B as potential biomarkers for prostate cancer.

AIM: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. MATERIALS & METHODS: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. RESULTS: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. CONCLUSION: These data support the strategy and the potential use of the biomarkers for PrCa.

Clinical Validation of a Serum Protein Panel (FLNA, FLNB and KRT19) for Diagnosis of Prostate Cancer.

This study reports on the development of a novel serum protein panel of three prostate cancer biomarkers, Filamin A, Filamin B and Keratin-19 (FLNA, FLNB and KRT19) using multivariate models for disease screening and prognosis. ELISA and IPMRM (LC-MS/MS) based assays were developed and analytically validated by quantitative measurements of the biomarkers in serum. Retrospectively collected and clinically annotated serum samples with PSA values and Gleason scores were analyzed from subjects who underwent prostate biopsy, and showed no evidence of cancer with or without indication of prostatic hyperplasia, or had a definitive pathology diagnosis of prostatic adenocarcinoma. Probit linear regression models were used to combine the analytes into score functions to address the following clinical questions: does the biomarker test augment PSA for population screening? Can aggressive disease be differentiated from lower risk disease, and can the panel discriminate between prostate cancer and benign prostate hyperplasia? Modelling of the data showed that the new prostate biomarkers and PSA in combination were better than PSA alone in identifying prostate cancer, improved the prediction of high and low risk disease, and improved prediction of cancer versus benign prostate hyperplasia.