Methods and Compositions for Selecting siRNA of Improved Functionality

Abstract

Efficient sequence specific gene silencing is possible through the use of siRNA technology. By selecting particular siRNAs by rational design, one can maximize the generation of an effective gene silencing reagent, as well as methods for silencing genes. Methods, compositions, and kits generated through rational design of siRNAs are disclosed.

1 - 13 . (canceled) 
     
     
         14 . A method for developing an siRNA algorithm for selecting siRNA, said method comprising:
 (a) selecting a set of siRNA;   (b) measuring gene silencing ability of each siRNA from said set;   (c) determining relative functionality of each siRNA;   (d) determining improved functionality based on the following variables: the presence or absence of a particular nucleotide at a particular position, the total number of As and Us in positions 15-19, the number of times that the same nucleotide repeats within a given sequence, and the total number of Gs and Cs; and   (e) developing an algorithm using the information of step (d).   
     
     
         15 . A method of selecting an siRNA with improved functionality, said method comprising using the algorithm of  claim 14 . 
     
     
         16 . (canceled) 
     
     
         17 . A method for identifying hyperfunctional siRNA, comprising: applying selection criteria to a set of potential siRNAs that comprise 18-30 base pairs, wherein said selection criteria are non-target specific criteria, and said set comprises at least two siRNAs and each of said at least two siRNAs contains a sequence that is at least substantially complementary to a target gene; and determining the relative functionality of the at least two siRNAs and assigning each of the at least two siRNAs a functionality score; and selecting siRNAs from the at least two siRNAs that have a functionality score that reflects greater than 80 percent silencing at a concentration in the picomolar range, wherein said greater than 80 percent silencing endures for greater than 120 hours. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A method according to  claim 14 , wherein said siRNA are unimolecular. 
     
     
         21 . (canceled) 
     
     
         22 . A method according to  claim 17 , wherein said siRNA are unimolecular. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . A method according to  claim 14 , wherein said siRNA are comprised of two separate polynucleotide strands. 
     
     
         26 . (canceled) 
     
     
         27 . A method according to  claim 17 , wherein said siRNA are comprised of two separate polynucleotide strands. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . A method according to  claim 14 , wherein said siRNA are expressed from one or more vectors. 
     
     
         31 . (canceled) 
     
     
         32 . A method according to  claim 17 , wherein said siRNA are expressed from one or more vectors. 
     
     
         33 - 36 . (canceled) 
     
     
         37 . A method according to  claim 17 , wherein two or more genes are silenced by a single administration of siRNA. 
     
     
         38 - 41 . (canceled) 
     
     
         42 . An RNA molecule capable of inducing the RNA interference pathway, wherein said RNA molecule consists of: (a) a duplex region; and (b) either no overhang regions or at least one overhang region, wherein each overhang region contains six or fewer nucleotides, wherein the duplex region consists of a sense region and an antisense region, wherein said sense region and said antisense region together form said duplex region and said sense region and said antisense region are each 19-30 nucleotides in length and said antisense region comprises a sequence that is the complement of a sequence selected from the group comprised of SEQ ID NO: 1 to 2675299. 
     
     
         43 . The RNA molecule of  claim 42 , wherein said antisense region and said sense region are each 19-25 nucleotides in length. 
     
     
         44 . The RNA molecule of  claim 43 , wherein said antisense region and said sense region are each 19 nucleotides in length. 
     
     
         45 . The RNA molecule of  claim 42 , wherein said RNA molecule has at least one overhang region. 
     
     
         46 . The RNA molecule of  claim 42 , wherein said RNA molecule has no overhang regions. 
     
     
         47 . A method for inhibiting the expression of a gene in a cell, the method comprising introducing into the cell in vitro the RNA molecule of  claim 42 . 
     
     
         48 . The method according to  claim 47 , wherein the cell is a human cell. 
     
     
         49 . The method according to  claim 47 , wherein said introducing is via transfection. 
     
     
         50 . The method according to  claim 47 , wherein said introducing is via passive uptake. 
     
     
         51 . A pool of at least two RNA molecules capable of inducing the RNA interference pathway, wherein said pool comprises a first RNA molecule and a second RNA molecule, wherein said first RNA molecule is the RNA molecule of  claim 42 , wherein the duplex region of said first RNA molecule is a first duplex region and said second RNA molecule consists of a second duplex region and either no overhang regions or at least one overhang region, wherein each overhang region contains six or fewer nucleotides, wherein the second duplex region of said second RNA molecule comprises a sense region and an antisense region, wherein said sense region and said antisense region of said second RNA together form said second duplex region and said sense region and said antisense region of said second duplex region are each 19-30 nucleotides in length and said antisense region of said second duplex region comprises a sequence that is the complement of a sequence selected from the group consisting of SEQ ID NO: 1 to 2675299.