Jinghua Yu

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice

Triantennary N-acetyl galactosamine (GalNAc, GN3), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2′-O-Et-2′,4′-bridged nucleic acid) gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.

Comprehensive Structure-Activity Relationship of Triantennary N-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.

Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes

Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

Synthesis and biological evaluation of sialyl-oligonucleotide conjugates targeting leukocyte B trans-membranal receptor CD22 as delivery agents for nucleic acid drugs.

Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.

Synthesis and Antisense Properties of 2′‐O‐(2S‐Methoxypropyl)‐RNA‐Modified Gapmer Antisense Oligonucleotides

To ascertain whether increasing hydrophobicity can enhance the activity of second‐generation antisense oligonucleotides (ASOs) in muscle, we investigated the antisense properties of 2′‐O‐(2S‐methoxypropyl)‐RNA (2S‐MOP)‐modified ASOs. Synthesis of the 2S‐MOP 5‐methyl uridine phosphoramidite was accomplished on a multi‐gram scale by Lewis‐acid‐catalyzed ring opening of 5′‐O‐tert‐butyldiphenylsilyl ether‐protected 2,2′‐anhydro‐5‐methyl uridine with 2S‐methoxy‐1‐propanol. Synthesis of the 2S‐MOP 5‐methyl cytidine nucleoside from the corresponding 5‐methyl uridine nucleoside was accomplished by formation and displacement of a 4‐triazolide intermediate with aqueous ammonia. 2S‐MOP‐modified oligonucleotides were prepared on an automated DNA synthesizer and showed similar enhancements in duplex thermal stability as 2′‐O‐methoxyethyl RNA (MOE)‐modified oligonucleotides. 2S‐MOP‐containing antisense oligonucleotides were evaluated in Balb‐c mice and showed good activity for decreasing the expression levels of scavenger receptor B1 (Srb1) and phosphatase and tensin homologue (PTEN) mRNA in liver and muscle tissue.