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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.

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.

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. ispartof: NUCLEIC ACIDS RESEARCH vol:48 issue:8 pages:4028-4040 ispartof: location:England status: published

We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.

The Thrombopoietin (THPO)/ thrombopoietin receptor (MPL) signaling axis is not only critical for the generation of platelets and megakaryocytes, but also for the maintenance of hematopoietic stem cells (HSC) and the bone marrow niche. MPL is expressed on primitive HSC, HSC progenitors, megakaryocytes, platelets, osteoblasts and osteoclasts, clonal hematopoietic stem cells and many leukemias. THPO production is constitutive but is also increased by inflammatory cytokines. Sustained exposure to high levels of THPO not only enhances platelet production, but also has a profound effect on HSC and the bone marrow microenvironment. Excess THPO/MPL signaling, whether driven by inflammatory cytokines, or due to mutations in THPO, JAK2, MPL or CALR, is associated with HSC expansion, megakaryocyte hypertrophy and increased platelet count, excess release of megakaryocyte and platelet-based cytokines such as TGF-beta and PDGF-alpha, and the development of stromal myofibroblasts that drive tissue fibrosis and anemia. We developed a robust and clinically validated RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent N-acetylgalactosamine (GalNAc) conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous injection. Since liver is the major source of THPO expression, we utilized GalNAc-siRNA technology to develop siRNAs targeting THPO for evaluation in wild type mice and murine models of myeloproliferative neoplasms (MPN). Active siRNAs were identified by in vitro screening in primary mouse hepatocytes and the 12 best siRNAs were evaluated in vivo in normal mice to select the most potent siRNA. THPO liver mRNA levels were reduced by up to 80% after a single subcutaneous THPO siRNA dose, with no effect on THPO mRNA expression in other organs (kidney, and bone marrow, both of which had marginal THPO expression compared to liver). Circulating TPO levels were reduced by 80% by day 7 and were suppressed for up to 28 days post a single dose treatment. Platelet counts were reduced to 60% of baseline by day 14, and a further reduction to more than 70% of baseline was observed with every other week dosing. No changes in red blood cell or white blood cell subsets were observed. Platelet reduction was accompanied by a reduction in megakaryocyte mass, as evidenced by a 50% decrease in the number of bone marrow megakaryocytes in THPO siRNA treated mice compared to controls. Mice treated every other week with TPO siRNA for three months demonstrated sustained circulating THPO protein and platelet count reductions, and a significant reduction in bone marrow HSC, Lin-Sca1+Kit- (LSK) and multipotent progenitor (MPP) frequency. Evaluation of impact THPO silencing on MPN phenotypes in transgenic JAK2V617F mice is ongoing. THPO silencing is a potential novel targeted therapeutic approach that may be beneficial in benign and malignant conditions in which deregulated THPO/MPL signal transduction drives disease pathology. Disclosures No relevant conflicts of interest to declare.