Generation and validation of structurally defined antibody-siRNA conjugates.

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes.

Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modification on siRNA.

Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair.

Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5'-C-Methyl Pyrimidine-Modified Oligonucleotides.

Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia.

siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.

Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed.

Metal-catalyzed 1,2-shift of diverse migrating groups in allenyl systems as a new paradigm toward densely functionalized heterocycles.

A general, mild, and efficient 1,2-migration/cycloisomerization methodology toward multisubstituted 3-thio-, seleno-, halo-, aryl-, and alkyl-furans and pyrroles, as well as fused heterocycles, valuable building blocks for synthetic chemistry, has been developed. Moreover, regiodivergent conditions have been identified for C-4 bromo- and thio-substituted allenones and alkynones for the assembly of regioisomeric 2-hetero substituted furans selectively. It was demonstrated that, depending on reaction conditions, ambident substrates can be selectively transformed into furan products, as well as undergo selective 6-exo-dig or Nazarov cyclizations.

Efficient synthesis of 2-mono- and 2,5-disubstituted furans via the CuI-catalyzed cycloisomerization of alkynyl ketones.

A mild, general, and efficient method for the synthesis of 2-monosubstituted and 2,5-disubstituted furans via the CuI-catalyzed cycloisomerization of alkynyl ketones was developed. It was demonstrated that furans containing both acid- and base-labile groups could be easily synthesized using this methodology. A plausible mechanism for this transformation is proposed.