Isis will make the following oral presentation at the ASGCT meeting to be held May 18-21, 2011 in Seattle, Washington
 Systemic Delivery of RNase H-Active Antisense Oligos in a Transgenic Mouse Model of Myotonic Dystrophy Type 1
Thurman M. Wheeler, Andrew J. Leger, Sanjay K. Pandey, A. Robert MacLeod, Masayuki Nakamori, Seng H. Cheng, C. Frank Bennett, Bruce M. Wentworth, Charles A. Thornton. Department of Neurology, University of Rochester, Rochester, NY; Genzyme Corporation, Framingham, MA; Isis Pharmaceuticals, Carlsbad, CA
Objective: To test whether systemic delivery of RNase H-active antisense oligos (ASOs) can reduce or eliminate RNA toxicity in a transgenic mouse model of myotonic dystrophy type 1 (DM1).
Background: DM1 is a dominantly inherited degenerative disease caused by expression of an expanded CUG repeat (CUGexp) in the 3′ UTR of the DMPK transcript. CUGexp RNA accumulates in the nucleus, sequesters poly(CUG) binding proteins, and forms nuclear inclusions. Trans-dominant effects of the mutant transcript include aberrant pre-mRNA splicing, dysregulated gene expression, myotonia, and muscular dystrophy. Human skeletal actin-long repeat (HSA-LR) transgenic mice express CUGexp RNA in the 3′ UTR of an hACTA1 transgene. In this model, the toxic RNA is retained in the nucleus and induces splicing changes similar to DM1. Antisense knockdown of pathogenic RNA would be expected to mitigate clinical features of DM1. However, skeletal muscle is less sensitive to ASO effects because distribution of ASOs to muscle tissue is low. In previous studies in rodents (n = 10), systemic ASOs failed to produce target knockdown in muscle, despite strong effects in liver.
Design/methods: ASOs were 2′ methoxyethyl gapmers that were designed to work by recruiting the nuclear enzyme RNase H to the ASO-RNA heteroduplex. ASOs targeting the hACTA1 mRNA were screened in cell culture. Active ASOs then were tested by subcutaneous injection in mice (25 mg/kg biweekly for 4 weeks). Control mice were treated with saline. Treatment assignments were randomized and analysis was blinded. Control ASOs targeted Malat1, Pten, and Srb1.
Results: Two hACTA1-targeting ASOs reduced transgene levels by up to 80%. Control ASOs had no effect. Serum chemistries and histopathology showed no evidence of toxicity. Knockdown of toxic RNA was associated with elimination of myotonia and correction of RNA mis-splicing in all muscles examined. These effects persisted at least 15 weeks after the final dose. By microarray analysis, > 85% of changes in gene expression were normalized or improved. Another nuclear-retained non-coding RNA, Malat1, was also sensitive to knockdown in muscle (up to 80%) using similar RNase H ASO designs. However, ASOs targeting endogenous mRNAs (Pten, Srb1) did not produce knockdown in muscle, and the distribution of ASOs to skeletal muscle in the DM1 model was not higher than in WT mice.
Conclusions: Systemic delivery of RNase H-active ASOs was surprisingly effective at reducing RNA toxicity in a transgenic mouse model of DM1. Nuclear-retained transcripts may display increased sensitivity to RNase H-active ASOs and enable RNA knockdown in muscle tissue where biodistribution is low. The mechanism may relate to residence time of transcripts in the nucleus, the cellular compartment in which RNase H1 is also located. ASOs that act through the RNase H pathway may exploit the nuclear retention phenomenon to gain a therapeutic advantage in DM1.
Keywords: Oligonucleotide Based Therapies; RNA; Genetic Diseases
Date: Thursday, May 19, 2011
Session Info: Simultaneous Oral Abstract Sessions: Small RNA and Oligonucleotide Based Therapeutics (2:30 PM-4:30 PM)
Presentation Time: 2:30 pm