ABSTRACT
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.
Subject(s)
Acetylgalactosamine/chemical synthesis , Acetylgalactosamine/pharmacology , Hepatocytes/drug effects , Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/pharmacology , Animals , Apolipoprotein C-III/drug effects , Drug Delivery Systems , Factor XI/drug effects , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Scavenger Receptors, Class B/biosynthesis , Scavenger Receptors, Class B/genetics , Structure-Activity RelationshipABSTRACT
Efficient synthesis of phosphorothioate RNA (PS-RNA) is demonstrated by using phenylacetyl disulfide (PADS) in a mixture of pyridine and acetonitrile (1:1, v/v) for 3 min. Sulfurization is achieved with >99.8% stepwise efficiency. This reagent also performs efficiently during synthesis of RNA containing PS:PO mixed backbone.
Subject(s)
Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/therapeutic use , Phenylacetates/chemistry , RNA, Small Interfering/chemical synthesis , RNA, Small Interfering/therapeutic use , Sulfides/chemistry , Thionucleotides/chemical synthesis , Thionucleotides/therapeutic use , Molecular Structure , Oligonucleotides, Antisense/chemistry , RNA, Small Interfering/chemistry , Thionucleotides/chemistryABSTRACT
Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.
