Human LNA Test
Reporting on advances in locked nucleic acid (LNA) development, Troels Koch, Ph.D., vp, research at Santaris Pharma, spoke at the conference regarding “Potent Single-Stranded Inhibition of Coding and Non-Coding RNA.”
Dr. Koch’s participation at the meeting came less than two weeks after his company announced that its LNA drug, SPC3649 (LNA-antimiR™-122) had become the first miRNA medicine to be tested in humans. The first clinical trial will involve 48 male volunteers.
“LNA’s high binding strength to its complementary target is an important component to the high potency, which in turn, allows significant reductions in dosage concentrations combined with good specificity,” Dr. Koch explained. Bio-stability is also much improved compared to benchmark phosphorothioate chemistry. LNA oligonucleotides can be designed as both gap-mers for targeting mRNA and mix-mers for targeting miRNA.
The former segments of LNA nucleotides are posted at either end of the DNA/PS chain, while a mix-mer—as the name implies—has LNA nucleotides interspersed in several locations. Biodistribution of a 16-mer gap-mer in mice 24 hours after a single intravenous dose was broad and included bone marrow (5%), kidney (21%), liver (11%), lymph node (14%), and spleen (4%).
Dr. Koch described a study in mice with a 16-mer LNA against ApoB-100 mRNA, the major apolipoprotein of low-density lipoproteins, which is responsible for carrying cholesterol to tissues. Mice dosed three consecutive days at 25 mg/kg showed an 80% reduction in ApoB mRNA and a 70% reduction in total plasma cholesterol.
The effect was of long duration, moderating to about 50% after approximately three weeks. Dr. Koch also reported a significant potency increase of ApoB mRNA down regulation in mouse liver going from the 16-mer to a 12-mer. The 12-mer showed significantly higher potency for ApoB mRNA down-regulation and the corresponding reductions in plasma cholesterol was also more efficacious. In fact, a total dose of 3 mg/kg was enough to reduce total plasma cholesterol by 50%.
Dr. Koch explained that the potency induction by LNA observed for mRNA down-regulations was also an important property for potent miRNA inhibition. The potency for miRNA inhibition of LNA mix-mers closely followed the affinity of the mix-mers for the mature miRNA. The most potent 15-mer, SPC3649, was 10–20 times more potent for miR122 inhibition compared to a 16-mer with 10 degrees lower Tm.
Dr. Koch showed that, following the inhibition of miR122 expression, total plasma cholesterol was reduced in mice and in nonhuman primates. In the monkey study, four groups of five African green monkeys were dosed at 1, 3, and 10 mg/kg levels on days 1, 3, and 5, while the fourth group acted as the control.
Relative plasma cholesterol reduction was dose-related generally with the lowest level reached in all three groups between day 20 and day 40. Liver biopsies at day 6 demonstrated localization of the LNA-antimiR in hepatocytes, and complete duplex formation between SPC3649 and miR122.
It was also shown that inhibition of miR122 by SPC3649 led to a potent inhibition of HCV replication in HUH7 hepatocytes. The latter has interest for HCV treatments since miR122 is a host factor and thus represents a potential new approach for treating HCV patients.
Finally, LNAs are generally well tolerated, with low acute toxicity compared to PS oligonucleotides, and with no clinically meaningful biochemical, hematologic, or histopathologic toxic effects in rodents or monkeys at clinically relevant doses.
As a result of the improved affinity, pharmacokinetics, and toxicity profiles, LNAs possesses attractive drug-like properties and a useful therapeutic index while providing siRNA-like potencies without the complications of complex delivery systems. The conclusion in the presentation was that LNA serves as a common platform for potent inhibition of both mRNA and microRNA.