The expanded CAG repeat in the huntingtin gene as target for therapeutic RNA modulation throughout the HD mouse brain.

The aim of these studies was to demonstrate the therapeutic capacity of an antisense oligonucleotide with the sequence (CUG)7 targeting the expanded CAG repeat in huntingtin (HTT) mRNA in vivo in the R6/2 N-terminal fragment and Q175 knock-in Huntington's disease (HD) mouse models. In a first study, R6/2 mice received six weekly intracerebroventricular infusions with a low and high dose of (CUG)7 and were sacrificed 2 weeks later. A 15-60% reduction of both soluble and aggregated mutant HTT protein was observed in striatum, hippocampus and cortex of (CUG)7-treated mice. This correction at the molecular level resulted in an improvement of performance in multiple motor tasks, increased whole brain and cortical volume, reduced levels of the gliosis marker myo-inositol, increased levels of the neuronal integrity marker N-aceyl aspartate and increased mRNA levels of the striatal marker Darpp-32. These neuroanatomical and neurochemical changes, together with the improved motor performance, suggest that treatment with (CUG)7 ameliorates basal ganglia dysfunction. The HTT-lowering was confirmed by an independent study in Q175 mice using a similar (CUG)7 AON dosing regimen, further demonstrating a lasting reduction of mutant HTT protein in striatum, hippocampus and cortex for up to 18 weeks post last infusion along with an increase in motor activity. Based on these encouraging results, (CUG)7 may thus offer an interesting alternative HTT-lowering strategy for HD.

Design and analysis of effects of triplet repeat oligonucleotides in cell models for myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2'-O-methyl (2'-OMe) phosphorothioate (PT)-modified (CAG)7 oligo (PS58), which selectively silences mutant DMPK transcripts through recognition of the abnormally long (CUG)n tract. We present here a comprehensive collection of triplet repeat AONs and found that oligo length and nucleotide chemistry are important determinants for activity. For significant reduction of expanded DMPK mRNAs, a minimal length of five triplets was required. 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified AONs appeared not effective, probably due to lack of nuclear internalization. Selectivity for products from the expanded DMPK allele in patient myoblasts, an important requirement to minimize unwanted side effects, appeared also dependent on AON chemistry. In particular, RNase-H-dependent (CAG)n AONs did not show (CUG)n length specificity. We provide evidence that degradation of long DMPK transcripts induced by PS58-type AONs is an RNase-H independent process, does not involve oligo-intrinsic RNase activity nor does it interfere with splicing of DMPK transcripts. Our collection of triplet repeat AONs forms an important resource for further development of a safe therapy for DM1 and other unstable microsatellite diseases.Molecular Therapy-Nucleic Acids (2013) 2, e81; doi:10.1038/mtna.2013.9; published online 19 March 2013.

Bioavailability of soil organic matter and microbial community dynamics upon permafrost thaw.

Amplified Arctic warming could thaw 25% of the permafrost area by 2100, exposing vast amounts of currently fixed organic carbon to microbially mediated decomposition and release of greenhouse gasses through soil organic matter (SOM) respiration. We performed time-series incubation experiments with Holocene permafrost soils at 4°C for up to 11 days to determine changes in exoenzyme activities (EEAs) (i.e. phosphatase, ß-glucosidase, aminopeptidase) as a measure for the bioavailability of SOM in response to permafrost thaw. We also profiled SSU rRNA transcripts to follow the qualitative and quantitative changes in viable prokaryotes and eukaryotes during incubation. EEA, amount of rRNA transcripts and microbial community structures differed substantially between the various soil intervals in response to thaw: after 11 days of incubation, the active layer became slightly depleted in C and P and harboured bacterial phyla indicative of more oligotrophic conditions (Acidobacteria). A fast response in phosphatase and ß-glucosidase upon thaw, and a predominance of active copiotrophic Bacteroidetes, showed that the upper permafrost plate serves as storage of easily degradable carbon derived from the overlying thawed active layer during summer. EEA profiles and microbial community dynamics furthermore suggest that the deeper and older permafrost intervals mainly contain recalcitrant SOM, and that extracellular soil-bound exoenzymes play a role in the initial cleavage of biopolymers, which could kick-start microbial growth upon thaw. Basidiomycetous fungi and Candidate Subdivision OP5 bacteria were the first to respond in freshly thawed deeper permafrost intervals, and might play an important role in the decomposition of recalcitrant SOM to release more labile substrates to support the major bacterial phyla (ß-Proteobacteria, Actinobacteria, Firmicutes), which predominated thereafter.