Reduced size self-delivering nucleic acid compounds targeting long non-coding RNA
Inventors
Cardia, James • Bulock, Karen G. • Vandesompele, Joke Hedwig • Van Peer, Gert
Assignees
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Publication Number
US-11021707-B2
Publication Date
2021-06-01
Expiration Date
Abstract
The present disclosure relates to RNAi constructs with improved cellular uptake characteristics and methods of use of these compounds for silencing expression of long coding RNAs (IncRNAs).
Core Innovation
The disclosure relates to isolated, double stranded nucleic acid molecules designed as lncRNA inhibitors. The molecules include a guide strand complementary to a lncRNA sequence and a shorter passenger strand, arranged as a double stranded region and a single stranded region at the 3′ end of the guide strand.
The lncRNA inhibitor is specifically described for inhibiting the lncRNA MALAT1. The disclosed sequence-listing content provides antisense/guide-strand oligonucleotides targeting lncRNAs including lncMALAT1/MALAT1 and additional lncRNAs such as lncFAM22E1, lncRala1, lncZBTB42, lncPANK1, lncEBF3, lncScand, lncFAM69C2, lncVEZF1, lncFBXO256, and lncNDST3.
The nucleic acid molecules incorporate hydrophobic conjugates and hydrophobic conjugated constructs, including cholesterol conjugates and other lipophilic group examples. The chemical backbone and linkage substitutions include phosphorothioate and related substitute linkages, as well as sugar modifications such as 2′-O-methyl, 2′-fluoro, and 2′-OH.
Claims Coverage
Two independent claims are provided across the input items: one directed to an isolated, double stranded nucleic acid molecule and one directed to a method of delivering such a molecule to a cell. The coverage is grounded in multiple inventive feature constraints, including defined guide/passenger architecture, MALAT1 inhibition, specified SEQ ID NO sequence inclusion, and constrained chemical modification requirements.
MALAT1-targeted double stranded lncRNA inhibitor architecture
An isolated, double stranded nucleic acid molecule comprising a guide strand of 18-23 nucleotides complementary to a lncRNA sequence and a passenger strand of 8-16 nucleotides, comprising a double stranded region and a single stranded region, wherein the single stranded region is the 3′ end of the guide strand and is 2-13 nucleotides in length.
Phosphorothioate and pyrimidine modification requirements
The isolated double stranded nucleic acid molecule comprises at least two phosphorothioate modifications, and at least 50% of the pyrimidines in the nucleic acid molecule are modified.
SEQ ID contiguous sequence constraint for MALAT1 inhibition
The isolated double stranded nucleic acid molecule is an lncRNA inhibitor of the lncRNA MALAT1 and comprises at least 12 contiguous nucleotides of a sequence selected from SEQ ID NOs: 287, 288, 293, 295, 299, 617, 618, 623, 625, and 629.
Hydrophobic conjugate delivery method for MALAT1 inhibition
A method of delivering a nucleic acid molecule to a cell comprising administering an isolated nucleic acid molecule to the cell, wherein the isolated nucleic acid molecule comprises a guide strand complementary to a lncRNA sequence and a passenger strand between 8-15 nucleotides in length, comprises at least two phosphorothioate modifications, at least 50% of the pyrimidines in the passenger strand are modified, and the molecule comprises a hydrophobic conjugate.
Overall, the independent claims require a MALAT1-targeted double stranded nucleic acid inhibitor with defined guide/passenger strand lengths and a specified 3′-end single stranded region, together with phosphorothioate modification and pyrimidine modification thresholds and a sequence constraint requiring at least 12 contiguous nucleotides from listed SEQ ID NOs. A hydrophobic conjugate is additionally required in the independent method claim for delivering the described nucleic acid to a cell.
Stated Advantages
Potent silencing of lncRNAs, including MALAT1, with screening outcomes reporting potent (>60% silencing) in vitro activity against multiple lncRNA targets.
Vehicle-free cellular uptake enabled by a hydrophobic/lipophilic conjugate.
Improved serum tolerance enabled by a hydrophobic/lipophilic conjugate.
The nucleic acid molecules incorporate hydrophobic conjugates and hydrophobic conjugated constructs to support delivery-related behavior.
The described constructs define nuclease resistance through non-hydrolizable phosphorothioate linkages.
Documented Applications
Modulating lncRNA expression and/or activity in cells by contacting the cell with the nucleic acid molecule in an amount effective to achieve the modulation.
In vivo/ex vivo use is described for modulating lncRNA expression/activity in connection with diseases linked to dysregulated lncRNAs.
Targeting and inhibiting the lncRNA MALAT1, including in human cancer cell lines where potent silencing is reported.
Inhibiting the lncRNA MALAT1.
Targeting lncRNAs including lncMALAT1/MALAT1, lncFAM22E1, lncRala1, lncZBTB42, lncPANK1, lncEBF3, lncScand, lncFAM69C2, lncVEZF1, lncFBXO256, and lncNDST3.
Delivering a nucleic acid molecule to a cell.
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