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Methods for identifying improved stereodefined phosphorothioate oligonucleotide variants of antisense oligonucleotides utilising sub-libraries of partially stereodefined oligonucleotides
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An antisense oligonucleotide and phosphorothioate technology, which is applied in nucleotide libraries, protein nucleotide libraries, libraries, etc., can solve the problems of lack of predictability
Inactive Publication Date: 2020-06-02
ROCHE INNOVATION CENT COPENHAGEN
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Embodiment 1
[0486] Synthesis of DNA 3'-O-oxazophosphine monomers was performed as previously described (Oka et al., J.Am.Chem.Soc. 2008 130:16031–16037; and Wan et al., NAR 2014, Nov. published online). Synthesis of LNA monomers was performed as previously described (WO2016 / 079181).
Embodiment 2
[0487] Example 2 Development of Sublibrary Discovery Method
[0488] Parent compound: 5'-G s m C s a s a s g s c s a s t s c s c s t s G s T-3' (SEQ ID NO 1), where capital letters represent β-D-oxylNA nucleosides (2'-O-CH2-4' bridged nucleosides in the β-D orientation), and small letters represent DNAnucleoside, subscript s represents a stereorandom phosphorothioate linkage, and m C is 5-methylcytosine.
[0489] Assaysystem: Oligonucleotides were tested in vitro at a concentration of 5 μM introduced into HeLa cells by stripped delivery. Cells were harvested after 3 days.
[0490] Analysis: Hif-1α mRNA knockdown was analyzed by qPCR.
[0491] The 13-mer parent compound has 12 phosphorothioate internucleoside linkages of undetermined stereochemistry. To identify sterically defined variants of the parent compound, two alternatives are utilized:
[0492] Strategy 1: Synthesis of 236 fully stereoconstrained compounds based on the parent compound wit...
Embodiment 3
[0501] Example 3: Examining the positional requirements of the 'rssr' motif
[0502] In Example 2, we determined that the most potent sub-library and few of the most potent compounds had a sterically defined internucleoside linkage "5'–RSSR 3'" motif, which was placed in turn at positions 5 and 6 The first Rp internucleoside bond between nucleosides is positioned together, referred to as position 5 ( Figure 10 shown in ). Data for the sublibrary for the position 5–8 region are provided below:
[0503]
[0504]
[0505] See data on fully stereodefined compounds from strategy 1 in the table below:
[0506] Position 5 (5-8 three-dimensional limited)
[0507]
[0508]
[0509] Position 6 (6–9 stereoscopically limited)
[0510]
[0511] We concluded that the position of the RSSR motif is critical for its effect on potency, and that subsequently moving the RSSR motif by 1 position, for example to position 6, generally results in a net loss of potency (also 11 sh...
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Abstract
The present invention relates to methods for identifying improved stereodefined phosphorothioate oligonucleotide variants of antisense oligonucleotides utilising sub-libraries of partially stereodefined oligonucleotides. The methods allow for the efficient identification of stereodefined variants with improved properties, such as enhancedin vitro or in vivo activity, enhanced efficacy, enhanced specific activity, reduced toxicity, altered biodistribution, enhanced cellular or tissue uptake, and / or enhanced target specificity (reduced off-target effects). Disclosed is a multiple parallel library screening approach where multiple exclusive or over-lapping short sub-regions or motifs of stereodefined phosphorothioate linked nucleosides are optimised to identify enhanced sub-libraries, and stereodefined internucleoside linkage patterns from each of the selected (improved) sub-libraries are then combined to produce an enhanced stereodefined compound.
Description
[0001] field of invention [0002] The present invention relates to methods of identifying improved sterically constrained phosphorothioate oligonucleotide variants of antisense oligonucleotides using partially sterically constrained oligonucleotide sublibraries. These methods allow efficient identification of sterically defined variants with improved properties, such as enhanced in vitro or in vivo activity, enhanced efficacy, enhanced specific activity, reduced toxicity, altered biodistribution, enhanced cellular or tissue uptake and / or Enhanced target specificity (reduced off-target effects). Background technique [0003] It has recently been established that the generation of stereorestricted phosphorothioate antisense oligonucleotide variants can be exploited to create superior pharmacological diversity and, as in the small moleculedrug discovery paradigm, the gap between stereodiastereomers Seemingly minor structural differences can yield compounds with major differenc...
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