Bi-functional short-hairpin RNA (bi-shRNA) specific for single-nucleotide KRAS mutations
Inventors
Rao, Donald • Wang, Zhaohui • Nemunaitis, John J. • Senzer, Neal
Assignees
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Publication Number
US-9353373-B2
Publication Date
2016-05-31
Expiration Date
Abstract
The present invention includes compositions and methods for making and using a bifunctional shRNAs capable of reducing an expression of a K-ras gene, e.g., a mutated K-ras gene, wherein at least one target site sequence of the bifunctional RNA molecule is located within the K-ras gene and wherein the bifunctional RNA molecule is capable of activating a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of K-ras.
Core Innovation
The invention relates to bifunctional shRNAs capable of reducing expression of a mutated K-ras gene. At least one target site sequence of the bifunctional RNA molecule is located within the K-ras gene, and the bifunctional RNA molecule activates a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of mutated K-ras while not reducing normal K-ras expression. The bifunctional shRNA comprises the sequence of SEQ ID NO: 56.
Mechanistically, the bifunctional shRNA activates both cleavage-dependent and cleavage-independent RNA-induced silencing complex pathways, producing siRNA-like and miRNA-like activities through RISC loading. The RISC activation is described with cleavage-dependent and cleavage-independent routes involving Argonaute proteins including Ago2 and additional Argonaute proteins (Ago1/2/3/4). The document describes an allele-selective approach that reduces mutated K-RAS expression without reducing wild-type KRAS expression, including assay-based evaluation such as a dual luciferase reporter with wild-type versus mutant K-RAS.
The disclosed compositions and constructs include a miR-30a backbone scaffold and a bifunctional stem-loop arrangement configured to create differential Ago engagement using guide strand versus passenger strand features and mismatch/bulge elements. The patent also describes sequence-targeting sets using human KRAS cDNA target sequences identified as SEQ ID NOS: 27-30, and bifunctional shRNA sequences identified as SEQ ID NOS: 1-22 and 31-56. Multitarget/multi-insert formats are disclosed, including triplet multimeric constructs using miR-17-92 cluster gap sequences for targeting specific KRAS mutations such as G12D, G12V, G12R, and G12C.
Claims Coverage
The claims cover bifunctional shRNA, expression vectors encoding the bifunctional shRNA for allele-selective inhibition of mutated K-ras, therapeutic delivery systems combining such vectors with a therapeutic agent carrier, and cells containing an expression unit producing the bifunctional shRNA. Across the independent claims, the main inventive content is organized into four inventive features.
Bifunctional shRNA enabling dual RISC activation for mutated K-ras knockdown without normal K-ras reduction
A bifunctional shRNA capable of reducing expression of a mutated K-ras gene, wherein at least one target site sequence is located within the K-ras gene and the bifunctional RNA molecule is capable of activating a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of mutated K-ras, without reducing normal K-ras expression, wherein the bifunctional shRNA comprises the sequence of SEQ ID NO: 56.
Expression vector encoding bifunctional shRNA for mutated K-ras inhibition via cleavage-dependent and cleavage-independent RNA-induced silencing
An expression vector comprising a promoter and a nucleic acid insert operably linked to the promoter, wherein the insert encodes one or more shRNA capable of inhibiting an expression of at least one target gene that is a mutated K-ras gene via RNA interference; wherein the one or more shRNA comprise a bifunctional RNA molecule that activates a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of the mutated K-ras, without reducing normal K-ras expression, wherein the bifunctional shRNA comprises the sequence of SEQ ID NO: 56.
Therapeutic delivery system combining a therapeutic agent carrier with an expression vector encoding bifunctional shRNA
A therapeutic delivery system comprising a therapeutic agent carrier and an expression vector comprising a promoter and a nucleic acid insert operably linked to the promoter, the nucleic acid insert encoding one or more short hairpin RNA capable inhibiting an expression of a target gene sequence that is mutated K-ras gene via RNA interference; wherein the one or more shRNA comprise a bifunctional RNA molecule that activates a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of the mutated K-ras, without reducing normal K-ras expression, wherein the bifunctional shRNA comprises the sequence of SEQ ID NO: 56.
Cell with an expression unit producing bifunctional shRNAs to reduce mutant K-ras expression without reducing normal K-ras expression
A cell comprising an expression unit that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 20, 21, 25, 50, 75, or 100 bifunctional shRNAs inserts capable of reducing an expression of a mutant K-ras gene; wherein at least one target site sequence of the bifunctional RNA molecule is located within the K-ras gene, wherein the bifunctional RNA molecule is capable of activating a cleavage-dependent and a cleavage-independent RNA-induced silencing complex for reducing the expression level of K-ras, without reducing normal K-ras expression, wherein the bifunctional shRNA comprises the sequence of SEQ ID NO: 56.
Across the independent claims, the patent requires a bifunctional shRNA sequence (SEQ ID NO: 56) that activates both cleavage-dependent and cleavage-independent RNA-induced silencing complexes to reduce expression of mutated K-ras while not reducing normal K-ras expression. Claim scope then expands this RNA concept into expression vectors, therapeutic delivery systems that include a therapeutic agent carrier, and cells containing expression units producing the bifunctional shRNAs.
Stated Advantages
Reduces expression level of mutated K-ras.
Does not reduce normal K-ras expression, enabling allele selectivity between mutated K-ras and normal K-ras.
Documented Applications
Therapeutic application to reduce mutated K-ras expression in pancreatic ductal adenocarcinoma (PDAC) and other KRAS-associated cancers.
Candidate drug evaluation methods based on mutant vs wild-type expression changes.
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