Gene Silencing by Single-Stranded Polynucleotides

Abstract

The present invention relates to compositions and methods for concurrently activating antisense and double-stranded RNase (dsRNase) mechanisms for inhibiting expression of a targeted gene, by delivering a single stranded bifunctional chimeric DNA/RNA oligonucleotide optimized for siRNA activity as well as antisense activity, into the nucleus of a target cell.

1 - 21 . (canceled) 
     
     
         22 . A method for preparing a single stranded polynucleotide for interfering with function of target RNA in a cell, comprising identifying a candidate functional double stranded siRNA to the target RNA; and synthesizing a single stranded polynucleotide sequence comprising at least a portion of the guidestrand of the candidate functional double stranded siRNA, wherein a functional single stranded polynucleotide which interferes with function of target RNA in a cell is formed, wherein the functional single stranded polynucleotide sequence comprises a 3′ RNA portion and a 5′ DNA portion, and wherein the DNA portion constitutes at least 50% of the functional single stranded polynucleotide. 
     
     
         23 . The method of  claim 22 , wherein the 3′ RNA portion comprises at least three consecutive ribonucleotides at the 3′ end. 
     
     
         24 . The method of  claim 22 , wherein the number of 3′ ribonucleotides of the single stranded polynucleotide is determined by assaying gene inhibition in cells plated on extracellular matrix protein. 
     
     
         25 . The method of  claim 24 , wherein the cells are on a tissue scaffold. 
     
     
         26 . The method of  claim 22 , wherein the number of 3′ ribonucleotides of the single stranded polynucleotide is determined by assaying gene inhibition in cells in multicellular spheroids. 
     
     
         27 . The method of  claim 22 , wherein the nucleotides of the single stranded polynucleotide are connected by phosphodiester bonds. 
     
     
         28 . The method of  claim 22 , wherein at least one of the nucleotides of the polynucleotide is a modified nucleotide. 
     
     
         29 . A method for mediating interference of target RNA in a cell, comprising:
 a. preparing a plurality of single stranded polynucleotides according to the method of  claim 22 ; and   b. delivering one or more of the single stranded polynucleotides in the absence of a passenger strand into said cell;   wherein the one or more of the delivered single stranded polynucleotides binds with Argonaute, wherein the bound single stranded polynucleotide and Argonaute activate dsRNase, resulting in interference of said target RNA.   
     
     
         30 . The method of  claim 29 , wherein Argonaute gene expression is present in the cell in an amount sufficient to activate said dsRNase. 
     
     
         31 . The method of  claim 29 , wherein the single stranded polynucleotide is formulated as a pharmaceutical composition comprising a non-viral, pharmaceutically acceptable carrier. 
     
     
         32 . The method of  claim 31 , wherein the composition also comprises a ligand that directs the composition to a targeted cell. 
     
     
         33 . The method of  claim 29 , wherein the step of delivering the single stranded polynucleotides into the cell comprises delivering the polynucleotides to the perinuclear region or the nucleus of the cell. 
     
     
         34 . The method of  claim 29 , wherein the cell is in a mammal, and the single stranded polynucleotide is delivered at a dose between about 1 attogram/kg body weight and about 100 ng/kg body weight. 
     
     
         35 . The method of  claim 29 , wherein one or more of the delivered single stranded polynucleotides also binds with target RNA to interfere with target RNA through antisense activity, wherein the duplex comprising said single stranded polynucleotide and said target RNA interferes with said target RNA through cleavage of said target RNA by RNase H enzyme activation, or by inhibition of transcription or translation of said target RNA by said binding, or a combination thereof. 
     
     
         36 . A method for mediating interference of target RNA in a cell, comprising:
 a. preparing a plurality of single stranded polynucleotides according to the method of  claim 22 ; and   b. delivering one or more of the single stranded polynucleotides in the absence of a passenger strand into said cell;   wherein the one or more of the delivered single stranded polynucleotides binds with target RNA, wherein the duplex comprising said single stranded polynucleotide and said target RNA interferes with said target RNA, said interference occurring through cleavage of said target RNA by RNase H enzyme activation, or by inhibition of transcription or translation of said target RNA by said binding, or a combination thereof.   
     
     
         37 . The method of  claim 36 , wherein RNAseH gene expression is present in the cell in an amount sufficient to activate RNAseH. 
     
     
         38 . The method of  claim 36 , wherein the step of delivering the polynucleotides into the cell comprises delivering the polynucleotides to the perinuclear region or the nucleus of the cell. 
     
     
         39 . A method of selecting a dosage level for administering a polynucleotide compound to a tissue for interfering with function of target RNA, comprising:
 a. characterizing said tissue for expression levels of Argonaute genes or RNAseH genes or both; and   b. selecting a dosage level for said polynucleotide compound based on said characterized expression level.   
     
     
         40 . A method for identifying a single stranded polynucleotide that interferes with function of target RNA in a cell, comprising: identifying one or more candidate functional double stranded siRNA's to the target RNA; synthesizing a single stranded polynucleotide sequence comprising at least a portion of the guidestrand of the candidate functional double stranded siRNA; and determining whether said candidate single stranded polynucleotide inhibits said target RNA, wherein the single stranded polynucleotide sequence comprises a 3′ RNA portion and a 5′ DNA portion, and wherein the DNA portion constitutes at least 50% of the functional single stranded polynucleotide.