Antisense oligonucleotides are now entering the clinic for hard-totreat diseases. New chemical modifications are urgently required to enhance their drug-like properties. We combine amide coupling with standard oligonucleotide synthesis to assemble backbone chimera gapmers that trigger an efficient RNase H response while improving serum life time and cellular uptake.Antisense oligonucleotides (ASOs) are short (B20mer) chemically modified oligomers that bind to their complementary RNA targets to modulate gene expression at the mRNA level. 1,2 Thus, ASOs can target proteins that are considered undruggable through conventional approaches. 3 As such, they hold enormous promise for hard-to-treat diseases as evidenced by a number of recently approved oligonucleotide (ON)-based drugs. [4][5][6] Chemical modifications are essential to improve the serum stability and pharmacodynamic properties of ASOs, as unmodified ONs are rapidly digested by nucleases in vivo 7,8 and suffer from poor cellular uptake and tissue distribution. 9 Whilst there have been considerable advances to modify ONs either at the nucleobase, sugar or backbone, a set of distinct chemical modifications to confer ideal drug-like properties has not yet been achieved. 10 Commonly used ribose modifications include 2 0 -F, 2 0 -OMe, 2 0 -O-(2-methoxyethyl) and locked nucleic acids, all of which have been shown to improve target affinity and serum stability. 11 The most commonly used phosphodiester (PO) mimic is the phosphorothioate (PS) linkage which is compatible with ribonuclease H (RNase H) activation, 12 a mechanism resulting in degradation of an mRNA upon formation of an ASO:mRNA heteroduplex. PS linkages also improve metabolic stability 13 and enhance pharmacodynamic properties through interactions with plasma proteins. 8,14,15 However, unspecific protein binding can contribute to the toxic potential of PS-ASOs 16,17 and the PS linkage is P-chiral resulting in a mixture of diastereomers (more than half a million isomers in Mipomersen 12,18 ). Moreover, inefficient cellular uptake remains a major challenge for ASO therapeutics. Therefore, the investigation of other artificial backbone linkages is urgently needed.Charge-neutral backbone modifications represent an interesting class of PO mimics. Among those, (thio)phosphonoacetate esters, 19 phosphotriesters 20 and alkyl phosphonates 21 can enhance cellular uptake by eliminating the PO negative charges. Moreover, incorporation of a single methylphosphonate can eliminate hepatotoxicity of PS-ASOs. 22 Phosphorodiamidate morpholino oligomers (PMOs) combine backbone and sugar modifications and enhance delivery through interactions with scavenger receptors, 23 but are not compatible with standard ON synthesis. All aforementioned PO mimics also suffer from increased steric complexity due to their P-chiral linkages.The absence of a chiral centre and the well-established solidphase peptide synthesis methods make the amide internucleoside linkage 24 (AM, Fig. 1A) a promising candidate for backbone surrogates. Within...