Bi-Functional Short-Hairpin RNA (Bi-shRNA) Specific for Single-Nucleotide KRAS Mutations

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.

1 . A bifunctional shRNA capable of reducing an expression of a 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. 
     
     
         2 . The bifunctional shRNAs of  claim 1 , wherein the bifunctional shRNA comprises at least one sequence defined by SEQ ID NOS: 1 to 22 or 31 to 56. 
     
     
         3 . (canceled) 
     
     
         4 . The bifunctional shRNAs of  claim 1 , wherein at least one target site sequence is within a human K-ras gene cDNA sequence (SEQ ID NOS: 27 to 30). 
     
     
         5 . The bifunctional shRNAs of  claim 1 , wherein the K-ras is a mutated K-ras. 
     
     
         6 . 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 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 K-ras.   
     
     
         7 . The expression vector of  claim 6 , wherein the target gene sequence comprises SEQ ID NO: 1 to 5. 
     
     
         8 . The expression vector of  claim 6 , wherein a sequence arrangement for the shRNA comprises a 5′ stem arm-19 nucleotide target, which is K-ras-TA-15 nucleotide loop-19 nucleotide target complementary sequence-3′stem arm-Spacer-5′ stem arm-19 nucleotide target variant-TA-15 nucleotide loop-19 nucleotide target complementary sequence-3′ stem arm. 
     
     
         9 . The expression vector of  claim 6 , wherein the nucleic acid insert comprises at least one sequence selected from SEQ ID NO: 6 to 27 or 53 to 56. 
     
     
         10 . The expression vector of  claim 6 , wherein the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 20, 21, 25, 50, 75, or 100 copies of bifunctional shRNAs inserts capable of reducing an expression of one or more mutated K-ras genes. 
     
     
         11 . The expression vector of  claim 6 , wherein at least one shRNA has a target site sequence that is within a mutated K-ras gene cDNA sequence. 
     
     
         12 . 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 (shRNA) capable inhibiting an expression of a target gene sequence that is 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 K-ras.   
     
     
         13 . The delivery system of  claim 12 , wherein the therapeutic agent carrier is a compacted DNA nanoparticle, or a compacted DNA nanoparticle with one or more polycations. 
     
     
         14 . The delivery system of  claim 13 , wherein the one or more polycations is a 10 kDA polyethylene glycol (PEG)-substituted cysteine-lysine 3-mer peptide (CK3oPEG10k). 
     
     
         15 . The delivery system of  claim 13 , wherein the compacted DNA nanoparticles are further encapsulated in at least one of a liposome, a reversibly masked liposome, or a bilamellar invaginated vesicle (BIV). 
     
     
         16 . The delivery system of  claim 12 , wherein the target gene sequence comprises SEQ ID NOS: 27 to 30. 
     
     
         17 . (canceled) 
     
     
         18 . The delivery system of  claim 12 , wherein the nucleic acid insert is selected from SEQ ID NOS: 1 to 22 or 31 to 56. 
     
     
         19 . A method to deliver one or more shRNAs to a target tissue expressing a K-ras gene comprising the steps of:
 preparing an expression vector comprising a promoter and a nucleic acid insert operably linked to the promoter that encodes the one or more shRNA, 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 K-ras;   combining the expression vector with a therapeutic agent carrier, wherein the therapeutic agent carrier comprises a liposome; and   administering a therapeutically effective amount of the expression vector and therapeutic agent carrier complex to a patient in need thereof.   
     
     
         20 . The method of  claim 19 , wherein the therapeutic agent carrier is a compacted DNA nanoparticle. 
     
     
         21 . The method of  claim 20 , wherein the DNA nanoparticle is compacted with one or more polycations, wherein the one or more polycations comprise a 10 kDA polyethylene glycol (PEG)-substituted cysteine-lysine 3-mer peptide (CK3oPEG10k) or a 30-mer lysine condensing peptide. 
     
     
         22 . The method of  claim 20 , wherein the compacted DNA nanoparticles are further encapsulated in a liposome, wherein the liposome is a bilamellar invaginated vesicle (BIV) that comprises one or more receptor targeting moieties. 
     
     
         23 . The method of  claim 22 , wherein the one or more receptor targeting moieties comprise small molecule bivalent beta-turn mimics. 
     
     
         24 . The method of  claim 19 , wherein the nucleic acid insert comprises a sequence selected from SEQ ID NOS: 1 to 22 or 53 to 56. 
     
     
         25 . A method to inhibit an expression of a K-ras gene in one or more target cells comprising the steps of:
 selecting the one or more target cells; and   transfecting the target cell with a vector that expresses one or more short hairpin RNA (shRNAs) capable of inhibiting an expression of a K-ras gene in the one or more target cells 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 K-ras.   
     
     
         26 . (canceled) 
     
     
         27 . A method of suppressing a tumor cell growth in a human subject comprising the steps of:
 identifying the human subject in need for suppression of the tumor cell growth; and   administering an expression vector in a therapeutic agent carrier complex to the human subject in an amount sufficient to suppress the tumor cell growth, wherein the expression vector expresses one or more shRNA capable inhibiting an expression of a target gene that is a mutated K-ras in the one or more target cells 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 target gene;   wherein the inhibition results in an apoptosis, an arrested proliferation, or a reduced invasiveness of the tumor cells.   
     
     
         28 - 48 . (canceled)