Polynucleotides for multivalent RNA interference, compositions and methods of use thereof
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Publication Number
US-9200276-B2
Publication Date
2015-12-01
Expiration Date
Abstract
The present invention includes bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and/or in which the target regions are complementary to more than one target gene or target sequence. Also included are compositions comprising such nucleic acid molecules and methods of using the same for multivalent RNA interference and the treatment of a variety of diseases and infections.
Core Innovation
The disclosure describes multivalent RNA interference agents comprising precisely structured polynucleotides with at most three target-specific regions. Each target-specific region is approximately 15–30 nucleotides in length and is fully or partially complementary to a target sequence, permitting each region to target multiple nucleotide sites within a single gene and/or multiple different genes by combining multiple target-specific regions in one structured agent.
One architecture is a polynucleotide complex comprising first, second, and third polynucleotides that hybridize via complementary 3′ and 5′ regions to form a polynucleotide complex with first, second, and third stem regions. The 3′ region of the first polynucleotide is complementary to a 5′ region of the second polynucleotide, the 3′ region of the second polynucleotide is complementary to a 5′ region of the third polynucleotide, and the 3′ region of the third polynucleotide is complementary to a 5′ region of the first polynucleotide.
A second architecture is a self-hybridizing polynucleotide consisting of a single polynucleotide molecule that includes first, second, and third target-specific regions. At least one of the target-specific regions binds or hybridizes to another target-specific region of the same molecule via complementary 3′ and 5′ regions to form at least one stem region and at least one loop or stem loop.
Claims Coverage
The provided material includes two independent claims. The inventive features focus on a three-polynucleotide, multistem polynucleotide complex with cyclic 3′/5′ complementarity and a single-molecule self-hybridizing architecture with three target-specific regions forming stem and loop or stem-loop structures.
Three-polynucleotide multistem polynucleotide complex with cyclic 3′/5′ complementarity
A polynucleotide complex consisting of a first, a second, and a third polynucleotide, each approximately 15–30 nucleotides in length, where the first polynucleotide is fully or partially complementary to a first target sequence, the second polynucleotide is fully or partially complementary to a second target sequence, and the third polynucleotide is either fully or partially complementary to a third target sequence or not specific to any target sequence; wherein a 3′ region of the first polynucleotide is complementary to a 5′ region of the second polynucleotide, a 3′ region of the second polynucleotide is complementary to a 5′ region of the third polynucleotide, and a 3′ region of the third polynucleotide is complementary to a 5′ region of the first polynucleotide; and wherein the first, second, and third polynucleotides hybridize via the complementary 3′ and 5′ regions to form a polynucleotide complex with first, second, and third stem regions.
Self-hybridizing single-polynucleotide with three target-specific regions forming stem and loop or stem-loop
A self-hybridizing polynucleotide consisting of a single polynucleotide molecule comprising a first, a second, and a third target-specific region, each approximately 15–30 nucleotides in length, where the first target-specific region is fully or partially complementary to a first target sequence, the second target-specific region is fully or partially complementary to a second target sequence, and the third target-specific region is either fully or partially complementary to a third target sequence or not specific to any target sequence; wherein a 3′ region of the first target-specific region is complementary to a 5′ region of the second target-specific region within the single polynucleotide molecule, a 3′ region of the second target-specific region is complementary to a 5′ region of the third target-specific region within the single polynucleotide molecule, and a 3′ region of the third target-specific region is complementary to a 5′ region of the first target-specific region within the single polynucleotide molecule; and wherein at least one of the first, second or third target-specific region binds or hybridizes to another target-specific region of the same single polynucleotide molecule via the complementary 3′ and 5′ regions to form at least one stem region and at least one loop or stem loop.
Overall, the claim coverage centers on multivalent RNA interference agents built from three target-specific regions (15–30 nucleotides each), arranged to create either a three-stranded polynucleotide complex with first, second, and third stem regions through cyclic 3′/5′ complementarity, or a single self-hybridizing polynucleotide that forms at least one stem region and at least one loop or stem loop.
Stated Advantages
Increased suppression versus shRNA controls for multivalent GFP suppression (trivalent Anti-GFP) when compared to controls.
Reduced activity upon strand deactivation for multivalent GFP suppression.
Substantially greater HIV replication inhibition for di-valent MV-siRNA targeting HIV gag and tat compared to gag-only.
Documented Applications
Reducing gene expression by introducing the specified polynucleotide complex, self-hybridizing polynucleotide, or a vector encoding such molecules into a cell.
Treating diseases and infections, including applications described in connection with HIV and ApoB targeting multivalent siRNAs.
Identifying gene function using the described multivalent RNA interference agents.
GFP suppression as an example application for the multivalent architectures.
HIV gag and tat targeting to inhibit HIV replication as an example application.
ApoB targeting (including trivalent/bivalent designs) for example applications.
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