Synthesis and Biophysical Properties of Constrained d‑Altritol Nucleic Acids (cANA)

The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how conformationally restricting the d-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found that the thermal stability was reduced (cANA/RNA −1.1 °C/modification) compared to DNA/RNA, suggesting the constrained system results in a small destabilizing perturbation in the duplex structure

Synthesis and Biophysical Properties of Constrained d‑Altritol Nucleic Acids (cANA)

The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how conformationally restricting the d-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found that the thermal stability was reduced (cANA/RNA −1.1 °C/modification) compared to DNA/RNA, suggesting the constrained system results in a small destabilizing perturbation in the duplex structure

Short Antisense Oligonucleotides with Novel 2′−4′ Conformationaly Restricted Nucleoside Analogues Show Improved Potency without Increased Toxicity in Animals

The potency of second generation antisense oligonucleotides (ASOs) in animals was increased 3- to 5 -fold (ED₅₀ ≈ 2−5 mg/kg) without producing hepatotoxicity, by reducing ASO length (20-mer to 14-mer) and by employing novel nucleoside modifications that combine structural elements of 2′-<i>O</i>-methoxyethyl residues and locked nucleic acid. The ability to achieve this level of potency without any formulation agents is remarkable and likely to have a significant impact on the future design of ASOs as therapeutic agents

Efficient Synthesis and Biological Evaluation of 5′-GalNAc Conjugated Antisense Oligonucleotides

Conjugation of triantennary <i>N</i>-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modified antisense oligonucleotides (5′-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3′-end of an ASO to the 5′-end results in improved potency in cells and animals

Comprehensive Structure–Activity Relationship of Triantennary <i>N</i>‑Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes

The comprehensive structure–activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc–ASO conjugates exhibited excellent potencies (ED₅₀ 0.5–2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc–ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs