A series of LNA antisense oligonucleotides of different length (10- to 20-mers) were designed with 100% sequence identity to the cynomolgus monkey and human apoB mRNA sequences. The 10- to 16-mers were designed to also have 100% sequence identity to mouse apoB mRNA. (Genbank accession no.: NM_000384 and NM_009693 for human and mouse apoB mRNA respectively; the cynomolgus mRNA was sequenced in-house). All oligonucleotides were designed as gap-mers containing 8–10 DNA nucleotides flanked by 1–5 LNA nucleotides at both ends with all internucleoside linkages phosphorothioated (Table 1).

In vitro properties of oligonucleotides

Oligonucleotides
OligonucleotideaLengthTm versus RNA (°C)IC50 in Huh-7 cells (nM)
5′-TTCAGcattggtattCAGTG-3′20-mer775.0 ± 1.2
5′-CAGcattggtatTCAg-3′16-mer632.7 ± 1.3
5′-CAGcattggtatTCA-3′15-mer600.5 ± 1.3
5′-AGCattggtatTCA-3′14-mer610.2 ± 1.2
5′-GCattggtatTCA-3′13-mer570.2 ± 1.3
5′-GCattggtatTC-3′12-mer530.4 ± 1.4
5′-CattggtatT-3′10-mer44ND
5′-gcattggtattc-3′12-mer PS34ND

aGap-mer oligonucleotides with LNA (capital) and DNA (plain font). All internucleoside linkages are phosphorothioated. Melting temperatures (Tm) of LNA oligonucleotides were measured against complementary RNA. ApoB mRNA (normalized to GAPDH) IC50 values were determined from three independent experiments ( ± SD). Non-detectable IC50 values, due to low potency, marked ND.

All oligonucleotides were synthesized using standard phosphoramidite protocols on an ÄKTA Oligopilot (GE Healthcare) at 130 µmol to 8 mmol scales employing custom made polystyrene primer supports. The DNA monomers were obtained from Proligo (Sigma-Aldrich) and the LNA monomers and solid support were produced by Santaris Pharma (commercially available from Exiqon, Denmark). After synthesis, the oligonucleotides were cleaved from the support using aqueous ammonia at 65°C overnight. The oligonucleotides were purified by ion exchange and desalted using a Millipore-membrane and were finally characterized by LC-MS (Reverse phase and ESI-MS).