Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals

Abstract

We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012.

Figure 1

Structures and duplex stabilizing properties of LNA, S-cEt, R-5′-Me-LNA, S-5′-Me-LNA, α-L-LNA, R-6′-Me-α-L-LNA, R-5′-Me-α-L-LNA, S-5′-Me-α-L-LNA, and 3′-Me-α-L-LNA in oligonucleotide sequences used for biophysical studies. LNA, locked nucleic acid.

Figure 2

Evaluation of LNA and α-L-LNA antisense oligonucleotides (ASOs) for hepatotoxicity in mice. Mice (n = 4/dose group) were injected intraperitoneally (i.p.) with a single dose of 35 mg/kg of ASOs A1 and A2 formulated in saline and animals were sacrificed 72 hours after injection. (a) Phosphatase and tensin homologue (PTEN) mRNA reduction in liver normalized to saline-treated animals and plasma alanine aminotransferase (ALT) levels post-sacrifice. (b) Sequence and T_m of ASOs A1 and A2 versus complementary RNA. Modified nucleotides are indicated in bold font, all errors in ±SD. LNA, locked nucleic acid.

Figure 3

Evaluation of LNA and α-L-LNA antisense oligonucleotides (ASOs) A3 and A4 for activity and hepatotoxicity in a single-dose–escalation study in mice. Mice (n = 4/dose group) were injected intraperitoneally (i.p.) with a single dose of 3.2, 10, 32, and 100 mg/kg of ASOs A3 and A4 formulated in saline and animals were sacrificed 72 hours after injection. (a) Phosphatase and tensin homologue (PTEN) mRNA reduction in liver normalized to saline-treated animals (b) plasma alanine aminotransferase (ALT) levels post-sacrifice. Percent change in (c) liver and (d) spleen weights relative to saline-treated animals. LNA, locked nucleic acid.

Figure 4

Evaluation of LNA A3, α-L-LNA A4, S-cEt A5, and R-6′-Me-α-L-LNA A6 for activity and hepatotoxicity in a 3-week dose-escalation study. Mice (n = 4/dose group) were injected intraperitoneally (i.p.) twice a week for 3 weeks with of 0.5, 1.5, 4.5, and 15 mg/kg of antisense oligonucleotides (ASOs) formulated in saline and animals were sacrificed 48 hours after last injection. ASOs A3, A4, and A5 were evaluated in one study while ASO A6 was evaluated in a parallel study using LNA ASO A3 as a control. Data for the LNA ASO (n = 8) reflects the average measurements from both studies while data for ASOs A4, A5, and A6 (n = 4) is from the individual studies (a) phosphatase and tensin homologue (PTEN) mRNA reduction in liver normalized to saline-treated animals (b) plasma alanine aminotransferase (ALT) levels post-sacrifice. Percent change in (c) liver and (d) spleen weights relative to saline-treated animals. All errors in ±SD.

Figure 5

Evaluation of 3′-Me-α-L-LNA A7, R-5′-Me-α-L-LNA A8, and S-5′-Me-LNA A9 for activity and hepatotoxicity in a 3-week dose-escalation study. Mice (n = 4/dose group) were injected intraperitoneally (i.p.) twice a week for 3 weeks with 2.5, 7.9, and 25 mg/kg of antisense oligonucleotides (ASOs) formulated in saline and animals were sacrificed 48 hours after last injection. (a) Phosphatase and tensin homologue (PTEN) mRNA reduction in liver normalized to saline-treated animals (b) plasma alanine aminotransferase (ALT) levels post-sacrifice. Percent change in (c) liver and (d) spleen weights relative to saline-treated animals. All errors in ±SD. LNA, locked nucleic acid.

Figure 6

Structural models of LNA and α-L-LNA dinucleotide units showing relative orientations of 3′-Me, 5′-Me, and 6′-Me groups. (a) Overlay of common structural units such as nucleobases, C1′ carbon and O4′ oxygen atoms of LNA (pink carbons) and α-L-LNA (teal carbons) dimers obtained from the NMR structures of modified DNA/RNA duplexes (refs. and 15). Complementary RNA strand not shown for clarity. (b) Structures of the monomeric units showing relative orientations of various methyl groups. (c) Relative orientations of S-6′-Me (light green), R-5′-Me (olive green), and S-5′-Me (dark blue) groups in a LNA dinucleotide unit. (d) Relative orientations of R-6′-Me (light green), R-5′-Me (olive green), S-5′-Me (dark blue), and 3′-Me (pink) groups in an α-L-LNA dinucleotide unit. LNA, locked nucleic acid.