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Isis Oligos Intervene in Coagulation Cascade

Inspired, perhaps, by their partnership with Archemix , Isis is testing a "sense" oligonucleotide to counteract the effects of an antisense inhibitor. Regado Biosciences, an Archemix licensee, is using a similar strategy with aptamers.

http://www.isispharm.com/featured_research_ASH-2007.html

>> Isis Pharmaceuticals Presents at the
American Society of Hematology 49th Annual Meeting Orlando, GA • December 8-11, 2007

Abstract Title:

Antisense Strategies to Specifically Deplete Levels of Factors II, VII and IX in Mice to Assess Their Potential as Targets for Anticoagulation

The ability to safely and effectively neutralize an anticoagulant’s activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) by allowing for less frequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from the kinetic study indicated that recovery of the anticoagulation established following 3 weeks of treatment with ISIS 401025 to PT-INR 2.5, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg ISIS 405277. When mice were pretreated with a different ASO targeting prothrombin, ISIS 405277 was unable to reverse either target depletion or anticoagulation indicating ISIS 405277’s neutralizing effects on ISIS 401025 are sequence specific. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.



Abstract:

A Sense Oligonucleotide Specifically Reserves the Prothrombin Depletion and Anticoagulation Caused by ISIS 401025, an Antisense Oligonucleotide Targeting Prothrombin RNA in Mice

Numerous potential intervention points exist within coagulation pathways for development of novel anticoagulant agents. To help determine the most suitable target for the discovery of novel antithrombotic agents, we have implemented an antisense strategy to specifically deplete levels of various coagulation factors in mice. Here we report the effects of antisense oligonucleotides (ASOs) targeting factors II, VII, and IX on target RNA and protein levels in mice, as well as PT/aPTT. ISIS 401025 targeting factor II, ISIS 403102 targeting factor VII, and ISIS 402618 targeting factor IX dose-dependently suppressed their respective target transcript levels in a specific manner in primary mouse hepatocytes. Following subcutaneous administration to mice, each ASO reduced target RNA levels in liver and target protein levels in plasma in a specific and dose-dependent fashion (ED95 of approximate 25 mg/kg for each ASO). In conjunction with these effects, clotting times were also prolonged in a similar dose responsive fashion. Both PT and aPTT were increased following factor II inhibition and a PT-INR of 2.5 was achieved at 25 mg/kg, corresponding to a reduction in prothrombin transcript of 95%. As expected following factor VII ASO treatment, PT was significantly increased, while aPTT was unchanged. Conversely, following factor IX ASO treatment, aPTT was marginally but significantly prolonged, while PT was unchanged. These results demonstrate the capability of ASOs to dose-dependently and specifically modulate coagulation factor activity depletion and support the ASO approach as a novel strategy for the discovery of novel anticoagulation agents. Studies are in progress to assess relative safety and to expand the anticoagulation profile in mice for each of the factors under study <<