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.

What is claimed is: 
     
         1 . A method for preparing a formulation comprising a single stranded polynucleotide for inhibiting gene expression in a mammalian cell, comprising:
 employing a suitable siRNA design algorithm to identify a candidate functional double stranded siRNA to a 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 consisting of 15-25 linked nucleosides without a self-complementary sequence region is formed; wherein the functional single stranded polynucleotide sequence consists of a 3′ RNA portion and a 5′ DNA portion where the RNA portion comprises at least three ribonucleotides and the DNA portion comprises at least 50% of said polynucleotide; wherein each of the internucleoside linkages is a phosphodiester linkage; and   formulating the resulting single stranded polynucleotide, in the absence of a passenger strand, with a pharmaceutically acceptable carrier for delivery to the perinuclear region or the nucleus of the mammalian cell.   
     
     
         2 . The method of  claim 1 , wherein the number of 3′ ribonucleotides of the single stranded polynucleotide is determined by assaying gene inhibition in cells plated on extracellular matrix protein. 
     
     
         3 . The method of  claim 2 , wherein the cells are on a tissue scaffold. 
     
     
         4 . The method of  claim 1 , wherein the number of 3′ ribonucleotides of the single stranded polynucleotide is determined by assaying gene inhibition in cells in multicellular spheroids. 
     
     
         5 . The method of  claim 1 , wherein at least one of the nucleotides of the polynucleotide is a modified nucleotide. 
     
     
         6 . The method of  claim 1 , wherein the pharmaceutically acceptable carrier is non-viral. 
     
     
         7 . The method of  claim 1 , wherein the pharmaceutically acceptable carrier further comprises a ligand that directs said carrier to the mammalian cell. 
     
     
         8 . A method of treating a patient having a disease characterized by the undesired production of a protein, comprising:
 a. determining the levels of Argonaute genes or RNAseH genes or both in a tissue sample of the patient; and   b. selecting, based on the level of Argonaute genes or RNAseH genes or both, a dosage level for administering a formulation prepared according to the method of  claim 1 ; and   c. administering the formulation to said patient.   
     
     
         9 . The method of  claim 8 , wherein said administration of the formulation concurrently activates antisense and dsRNAse mechanisms. 
     
     
         10 . The method of  claim 8 , wherein the disease is cancer. 
     
     
         11 . 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 siRNAs 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.