In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Hysteroscopic female sterilization with Essure in an outpatient setting.

The aim of this study is to evaluate the short and long-term results of hysteroscopic sterilization in an outpatient setting. Sixty-one women underwent hysteroscopic sterilization. At follow-up, all of the women were asked to complete a questionnaire concerning possible pregnancy, bleeding patterns, side-effects, or need for further therapy after sterilization. Technical feasibility, complications, patient satisfaction, and tubal occlusion based on X-ray or ultrasound were measured. Fifty-eight (95%) women were sterilized according to this method. Successful bilateral device placement was achieved in 52 women (85%) during the first attempt and in six (9.8%) during the second. A total of 50 (81.9%) women submitted completed outcome questionnaires. The mean follow-up period was 23 (range 7-67) months. No pregnancies were reported. All questionnaire respondents expressed overall satisfaction with the procedure. To conclude, Essure sterilization is a safe effective method for female sterilization that is feasible in the outpatient setting.