Multitargeting interfering RNAs having two active strands and methods for their design and use

Abstract

Interfering RNA molecules are now designed and produced with specificity for multiple binding sequences present in distinct genetic contexts in one or more pre-selected target RNA molecules and are used to modulate expression of the target sequences. The multitargeting interfering RNA molecules have two strands that target multiple target sites on one or more pre-selected RNA molecules. Such a multitargeting interfering RNA approach provides a powerful tool for gene regulation.

1 . A multitargeting interfering RNA molecule comprising Formula (I):  
         5′-p-XSY-3′ 3′-X′S′Y′-p-5′ wherein p consists of a terminal phosphate group that is independently present or absent;    wherein S consists of a first nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S′ consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S′ are at least substantially complementary to each other but are not palindromic;    wherein X, X′, Y, or Y′, is independently absent or consists of a nucleotide sequence;    wherein XSY is at least partially complementary to the first binding sequence to allow stable interaction therewith;    wherein Y′S′X′ is at least partially complementary to the second binding sequence to allow stable interaction therewith and is at least partially complementary to XSY to form a stable duplex therewith.    
     
     
         2 . The multitargeting interfering RNA molecule of  claim 1 , wherein X, X′, Y, or Y′, independently consists of one or more nucleotides.  
     
     
         3 . The multitargeting interfering RNA molecule of  claim 1 , wherein X consists of a third nucleotide sequence that is at least partially complementary to a second portion of the first binding sequence, said second portion is adjacent to and connected with the 3′-end of said first portion of the first binding sequence, and wherein X′ consists of a fourth nucleotide sequence that is substantially complementary to the third nucleotide sequence.  
     
     
         4 . The multitargeting interfering RNA molecule of  claim 3 , wherein X and X′ are completely complementary to each other.  
     
     
         5 . The multitargeting interfering RNA molecule of  claim 3 , wherein X is completely complementary to the second portion of the first binding sequence.  
     
     
         6 . The multitargeting interfering RNA molecule of  claim 1 , wherein Y′ consists of a fifth nucleotide sequence that is at least partially complementary to a second portion of the second binding sequence, said second portion is adjacent to and connected with the 3′-end of said first portion of the second binding sequence, and wherein Y consists of a sixth nucleotide sequence that is substantially complementary to the fifth nucleotide sequence.  
     
     
         7 . The multitargeting interfering RNA molecule of  claim 6 , wherein Y and Y′ are completely complementary to each other.  
     
     
         8 . The multitargeting interfering RNA molecule of  claim 6 , wherein Y′ is completely complementary to the second portion of the second binding sequence.  
     
     
         9 . The multitargeting interfering RNA molecule of  claim 1 , wherein S and S′ are completely complementary to each other.  
     
     
         10 . The multitargeting interfering RNA molecule of  claim 1 , wherein XS is completely complementary to the first portion and the second portion of the first binding sequence.  
     
     
         11 . The multitargeting interfering RNA molecule of  claim 1 , wherein Y′S′ is completely complementary to the first portion and the second portion of the second binding sequence.  
     
     
         12 . The multitargeting interfering RNA molecule of  claim 1 , wherein XSY and Y‘S’X′ are completely complementary to each other.  
     
     
         13 . The multitargeting interfering RNA molecule of  claim 1 , wherein S consists of a first nucleotide sequence of a length of about 8 to about 15 nucleotides.  
     
     
         14 . The multitargeting interfering RNA molecule of  claim 1 , wherein each of XSY and Y′S′X′ is of a length of about 15 to about 29 nucleotides.  
     
     
         15 . The multitargeting interfering RNA molecule of  claim 1 , wherein each of XSY and Y′S′X′ is of a length of about 19 to about 23 nucleotides.  
     
     
         16 . The multitargeting interfering RNA molecule of  claim 1  comprising one or more terminal overhangs.  
     
     
         17 . The multitargeting interfering RNA molecule of  claim 16 , wherein the overhang consists of 1 to 5 nucleotides.  
     
     
         18 . The multitargeting interfering RNA molecule of  claim 1  comprising at least one modified ribonucleotide, universal base, acyclic nucleotide, abasic nucleotide or non-ribonucleotide.  
     
     
         19 . The multitargeting interfering RNA molecule of  claim 18  comprising at least one 2′-O-methyl ribosyl substitution or a locked nucleic acid ribonucleotide.  
     
     
         20 . The multitargeting interfering RNA molecule of  claim 1 , wherein the first and the second binding sequences are present in distinct genetic contexts in one pre-selected target RNA molecule.  
     
     
         21 . The multitargeting interfering RNA molecule of  claim 1 , wherein the first and the second binding sequences are present in distinct genetic contexts in at least two pre-selected target RNA molecules.  
     
     
         22 . The multitargeting interfering RNA molecule of  claim 1 , wherein at least one of the pre-selected target RNA molecules is a non-coding RNA molecule.  
     
     
         23 . The multitargeting interfering RNA molecule of  claim 1 , wherein at least one of the pre-selected target RNA molecules is a mRNA molecule.  
     
     
         24 . The multitargeting interfering RNA molecule of  claim 1 , wherein at least one of the binding sequences is present in the 3′-untranslated region (3′UTR) of a mRNA molecule.  
     
     
         25 . The multitargeting interfering RNA molecule of  claim 1 , wherein one or more of the pre-selected target RNA molecules are involved in a disease or disorder of a biological system.  
     
     
         26 . The multitargeting interfering RNA molecule of  claim 25 , wherein one or more of the pre-selected target RNA molecules are involved in a disease or disorder of an animal or a plant.  
     
     
         27 . The multitargeting interfering RNA molecule of  claim 26 , wherein the animal is selected from the group consisting of a rat, a mouse, a dog, a cat, a pig, a monkey, and a human.  
     
     
         28 . The multitargeting interfering RNA molecule of  claim 26 , wherein one or more of the pre-selected target RNA molecules encode a protein of a class selected from the group consisting of receptors, cytokines, transcription factors, regulatory proteins, signaling proteins, cytoskeletal proteins, transporters, enzymes, hormones, and antigens.  
     
     
         29 . The multitargeting interfering RNA molecule of  claim 28 , wherein one or more of the pre-selected target RNA molecules encode a protein selected from the group consisting of ICAM-1, VEGF-A, MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1, bFGF, PlGF, VEGF-C, VEGF-D, β-catenin, κ-ras-B, κ-ras-A, EGFR, and TNF alpha.  
     
     
         30 . The multitargeting interfering RNA molecule of  claim 29  that decreases expression of any combination of ICAM-1, VEGF-B, VEGF-C, VEGF-D, IL-8, bFGF, PlGF, MCP-1 and IGF-1 in an expression system.  
     
     
         31 . The multitargeting interfering RNA molecule of  claim 29  that decreases expression of any combination of ICAM-1, VEGF-A and IGF-1 in an expression system.  
     
     
         32 . The multitargeting interfering RNA molecule of  claim 29  that decreases expression of both ICAM-1 and VEGF-A in an expression system.  
     
     
         33 . The multitargeting interfering RNA molecule of  claim 29  that decreases expression of any combination of β-catenin, K-ras, and EGFR in an expression system.  
     
     
         34 . The multitargeting interfering RNA molecule of  claim 29  that decreases expression of both Gluc6p and Inppl1 in an expression system.  
     
     
         35 . The multitargeting interfering RNA molecule of  claim 1 , wherein one or more of the pre-selected target RNA molecules encode a viral RNA.  
     
     
         36 . The multitargeting interfering RNA molecule of  claim 35 , wherein the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an influenza virus, a rhinovirus, and a severe acute respiratory syndrome (SARS) virus.  
     
     
         37 . The multitargeting interfering RNA molecule of  claim 36 , wherein the virus is a hepatitis C virus (HCV) and a pre-selected target RNA molecules encodes TNFalpha.  
     
     
         38 . The multitargeting interfering RNA molecule of  claim 1 , wherein one or more of the pre-selected target RNA molecules comprises one or more RNA molecules selected from a first biological system.  
     
     
         39 . The multitargeting interfering RNA molecule of  claim 1 , wherein one or more of the pre-selected target RNA molecules comprises one or more RNA molecules selected from a second biological system that is infectious to a first biological system.  
     
     
         40 . The multitargeting interfering RNA molecule of  claim 1 , wherein the pre-selected target RNA molecules comprise one or more RNA molecules selected from a first biological system and one or more pre-selected target RNA molecules selected from a second biological system that is infectious to the first biological system.  
     
     
         41 . The multitargeting interfering RNA molecule of  claim 40 , wherein the pre-selected target RNA molecules comprise one or more RNA molecules selected from an animal or a plant and one or more RNA molecules selected from a microbe or a virus that is infectious to the animal or the plant.  
     
     
         42 . The multitargeting interfering RNA of  claim 41 , wherein the pre-selected target RNA molecules comprises an RNA molecule encoding a human protein TNFalpha, LEDGF(p75), BAF, CCR5, CXCR4, furin, NFkB, STAT1.  
     
     
         43 . The multitargeting interfering RNA molecule of  claim 1 , wherein S consists essentially of a nucleotide sequence selected from the group consisting of: GUGACAGUCACU (SEQ ID NO: 2), CUGGGCGAGGCAG (SEQ ID NO: 21), GUGGAUGUGGAG (SEQ ID NO: 22), AGAATCGCAAAACCAGC (SEQ ID NO: 34), AGAATCGCAAAACCA (SEQ ID NO: 36), CAGGGGAGU (SEQ ID NO: 46), AGGGCUCCAGGCG (SEQ ID NO: 63) and GCUGGCCGAGGAG. (SEQ ID NO: 64)  
     
     
         44 . The multitargeting interfering RNA molecule of  claim 1 , wherein S′ consists essentially of a nucleotide sequence selected from the group consisting of: AGTGACTGTCAC (SEQ ID NO: 1), CUGCCUCGCCCAG (SEQ ID NO: 19), CUCCACAUCCAC (SEQ ID NO: 20), GCTGGTTTTGCGATTCT (SEQ ID NO: 33), TGGTTTTGCGATTCT (SEQ ID NO: 35), ACTCCCCTG (SEQ ID NO: 41), CGCCTGGAGCCCT (SEQ ID NO: 61) and CTCCTCGGCCAGC. (SEQ ID NO: 62)  
     
     
         45 . The multitargeting interfering RNA molecule of  claim 1 , consisting essentially of:  
       
         
           
                 
                 
                 
               
                     
                 
                   5′-CGAGUGACAGUCACUAGCUCC-3′ 
                   (SEQ ID NO: 3) 
                     
                 
                   3′-UAGCUCACUGUCAGUGAUCGA-5′; 
                   (SEQ ID NO: 4) 
                 
                     
                 
                   5′-UCGAGUGACAGUCACUAGCUC-3′ 
                   (SEQ ID NO: 7) 
                 
                   3′-CUAGCUCACUGUCAGUGAUCG-5′; 
                   (SEQ ID NO: 8) 
                 
                     
                 
                   5′-UCGAGUGACAGUCACUAGCUCC-3′ 
                   (SEQ ID NO: 11) 
                 
                   3′-CUAGCUCACUGUCAGUGAUCGA-5′; 
                   (SEQ ID NO: 12) 
                 
                     
                 
                   5′-CGAGUGACAGUCACUAGCUCC-3′ 
                   (SEQ ID NO: 3) 
                 
                   3′-UAGUUCACUGUCAGUGAUCGA-5′; 
                   (SEQ ID NO: 14) 
                 
                     
                 
                   5′-UCGAGUGACAGUCACUAGUUC-3′ 
                   (SEQ ID NO: 15) 
                 
                   3′-CUAGCUCACUGUCAGUGAUCG-5′; 
                   (SEQ ID NO: 8) 
                 
                     
                 
                   5′-CGAGUGACAGUCACUGAUUCC-3′ 
                   (SEQ ID NO: 16) 
                 
                   3′-CUAGCCACUGUCAGUGAUCGA-5′; 
                   (SEQ ID NO: 17) 
                 
                     
                 
                   5′-GAUCGAGUGACAGUCACUAGCUC-3′ 
                   (SEQ ID NO: 65) 
                 
                   3′-CUAGCUCACUGUCAGUGAUCG-5′; 
                   (SEQ ID NO: 8) 
                 
                     
                 
                   5′-CCUCACAGGGGAGUUGUGCCC-3′ 
                   (SEQ ID NO: 57) 
                 
                   3′-UCGGAGUGUCCCCUCAACACG-5′; 
                   (SEQ ID NO: 58) 
                 
                   and 
                 
                     
                 
                   5′-CCUCACAGGGGAGUUGUGCUU-3′ 
                   (SEQ ID NO: 59) 
                 
                   3′-UUGGAGUGUCCCCUCAACACG-5′; 
                   (SEQ ID NO: 60) 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         46 . A biological system comprising a multitargeting interfering RNA molecule comprising Formula (I):  
         5′-p-XSY-3′ 3′-X′S′Y′-p-5′ wherein p consists of a terminal phosphate group that is independently present or absent;    wherein S consists of a first nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S′ consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S′ are at least substantially complementary to each other but are not palindromic;    wherein X, X′, Y, or Y′, is independently absent or consists of a nucleotide sequence;    wherein XSY is at least partially complementary to the first binding sequence to allow stable interaction therewith;    wherein Y′S′X′ is at least partially complementary to the second binding sequence to allow stable interaction therewith and is at least partially complementary to XSY to form a stable duplex therewith.    
     
     
         47 . The biological system of  claim 46  being a virus, a microbe, a cell, a plant, or an animal.  
     
     
         48 . A vector comprising a nucleotide sequence that encodes the multitargeting interfering RNA molecule comprising Formula (I):  
         5′-p-XSY-3′ 3′-X′S′Y′-p-5′ wherein p consists of a terminal phosphate group that is independently present or absent;    wherein S consists of a first nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S′ consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S′ are at least substantially complementary to each other but are not palindromic;    wherein X, X′, Y, or Y′, is independently absent or consists of a nucleotide sequence;    wherein XSY is at least partially complementary to the first binding sequence to allow stable interaction therewith;    wherein Y′S′X′ is at least partially complementary to the second binding sequence to allow stable interaction therewith and is at least partially complementary to XSY to form a stable duplex therewith.    
     
     
         49 . The vector of  claim 48  being a viral vector.  
     
     
         50 . The vector of  claim 49  that is derived from a virus selected from the group consisting of an adeno-associated virus, a retrovirus, an adenovirus, a lentivirus, and an alphavirus.  
     
     
         51 . A cell comprising a vector wherein the vector comprises a nucleotide sequence that encodes the multitargeting interfering RNA molecule comprising Formula (I):  
         5′-p-XSY-3′ 3′-X′S′Y′-p-5′ wherein p consists of a terminal phosphate group that is independently present or absent;    wherein S consists of a first nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S′ consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S′ are at least substantially complementary to each other but are not palindromic;    wherein X, X′, Y, or Y′, is independently absent or consists of a nucleotide sequence;    wherein XSY is at least partially complementary to the first binding sequence to allow stable interaction therewith;    wherein Y′S′X′ is at least partially complementary to the second binding sequence to allow stable interaction therewith and is at least partially complementary to XSY to form a stable duplex therewith.    
     
     
         52 . The multitargeting interfering RNA molecule of  claim 1  wherein the molecule is a short hairpin RNA molecule.  
     
     
         53 . A vector for a short hairpin RNA molecule wherein the short hairpin RNA molecule is a multitargeting interfering RNA comprising Formula (I):  
         5′-p-XSY-3′ 3′-X′S′Y′-p-5′ wherein p consists of a terminal phosphate group that is independently present or absent;    wherein at least one of the terminal phosphate groups is absent and the strand XSY is linked to the strand Y′S′X′ 3′ to 5′ or 5′ to 3′ via a linker    wherein S consists of a first nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S′ consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S′ are at least substantially complementary to each other but are not palindromic;    wherein X, X′, Y, or Y′, is independently absent or consists of a nucleotide sequence;    wherein XSY is at least partially complementary to the first binding sequence to allow stable interaction therewith;    wherein Y′S′X′ is at least partially complementary to the second binding sequence to allow stable interaction therewith and is at least partially complementary to XSY to form a stable duplex therewith.    
     
     
         54 . A cell comprising the vector of  claim 53 .  
     
     
         55 . A pharmaceutical composition comprising a multitargeting interfering RNA molecule of  claim 1  and an acceptable carrier.  
     
     
         56 . A pharmaceutical composition comprising a vector of  claim 48  and an acceptable carrier.  
     
     
         57 . A pharmaceutical composition comprising a vector of  claim 53  and an acceptable carrier.  
     
     
         58 . A method of inducing RNA interference in a biological system, comprising the step of introducing a multitargeting interfering RNA molecule of  claim 1  into the biological system wherein the RNA molecule contacts target RNA and inhibits target RNA activity.  
     
     
         59 . A method for designing a multitargeting interfering RNA molecule, comprising the steps of: 
 a) selecting one or more target RNA molecules, wherein the modulation in expression of the target RNA molecules is desired;    b) obtaining at least one nucleotide sequence for each of the target RNA molecules;    c) selecting a length, n, in nucleotides, for a seed sequence, wherein n=about 6 or more;    d) obtaining a collection of candidate seeds of the length n from each nucleotide sequence obtained in step b), wherein a candidate seed and its complete complement are not palindromic, and the candidate seed occurs at least once in one or more of the nucleotide sequences obtained in step b), and its complete complement occurs at least once in one or more of the nucleotide sequences obtained in step b);    e) determining the genetic context of each of the candidate seed and its complete complement, by collecting, for each occurrence of the candidate seed and its complete complement, a desired amount of the 5′ and 3′ flanking sequence;    f) selecting a seed of the length n from the group of candidate seed;    g) selecting a first consensus target sequence, which comprises the seed and a consensus 3′-flanking sequence to the seed determined from the sequences obtained in step b);    h) selecting a second consensus target sequence, which comprises the complete complement of the seed and a consensus 3′-flanking sequence to the complete complement of the seed determined from the sequences obtained in step b);    i) obtaining a first strand sequence, which comprises the first consensus target sequence selected in step g) and, adjacent to and connected with the 5′-end of the first consensus target sequence, a complement of the consensus 3′ flanking sequence of step h);    j) obtaining a second strand sequence which comprises the second consensus target sequence selected in step h) and, adjacent to and connected with the 5′-end of the second consensus target sequence, a complement of the consensus 3′ flanking sequence of step g), and;    k) designing a multitargeting interfering RNA molecule comprising a first strand having the first strand sequence in step i) and a second strand having the second strand sequence obtained in step j).    
     
     
         60 . The method of  claim 59  wherein the step of obtaining a collection of candidate seeds of the length n comprises the steps of: 
 i) generating a first collection of sequences of the length n from each of the nucleotide sequences obtained in step b) of  claim 59 , using a method comprising the steps of: 
 1) beginning at a terminus of each of the nucleotide sequence;  
 2) sequentially observing the nucleotide sequence using a window size of n; and  
 3) stepping along the nucleotide sequence with a step size of 1;  
   ii) generating a second collection of sequences each of which is completely complementary to a sequence in the first collection; and    iii) obtaining the collection of candidate seeds of the length n from the inspection of the first and the second collections of sequences, wherein a candidate seed and its complete complement are not palindromic, and each candidate seed and its complete complement occurs at least once in the nucleotide sequences obtained in step b) of  claim 59 .    
     
     
         61 . The method of  claim 59  wherein the step of obtaining a collection of candidate seeds of the length n comprises the steps of: 
 i) obtaining the completely complementary sequence for each nucleotide sequence obtained in step (b) of  claim 59;     ii) generating a first collection of sequences of the length n from each of the nucleotide sequences obtained in step b) of  claim 59  and a second collection of sequences of the length n from each of the completely complementary sequences obtained in step (i), using a method comprising the steps of: 
 1) beginning at a terminus of the nucleotide sequence of each of the nucleotide sequences obtained in step b) of  claim 59  or each of the completely complementary sequences obtained in step (i);  
 2) sequentially observing the nucleotide sequence using a window size of n; and  
 3) stepping along the nucleotide sequence with a step size of 1; and  
   iii) obtaining the collection of candidate seeds of the length n from the inspection of the first and the second collections of sequences, wherein a candidate seed and its complete complement are not palindromic, and each of the candidate seeds is present in both the first and the second collections of sequences.    
     
     
         62 . The method of  claim 59 , wherein the step of selecting a group of candidate seeds comprises the step of discarding any sequence of the length n that 
 i) is composed of a consecutive string of 5 or more identical single nucleotides;    ii) is composed of only adenosine and uracil;    iii) is predicted to occur with unacceptable high frequency in the non-target transcriptome of interest;    iv) is predicted to have a propensity to undesirably modulate the expression or activity of one or more cellular component;    v) is any combination of i) to iv); or    vi) is palindromic.    
     
     
         63 . The method of  claim 59 , wherein each of the steps of selecting a first and a second consensus target sequence comprises the step of discarding any sequence that 
 i) is composed of only a single base;    ii) is composed of only adenosine and uracil;    iii) has a consecutive string of five or more bases which are cytosine;    iv) is predicted to occur with unacceptable high frequency in the non-target transcriptome of interest;    v) is predicted to have a propensity to undesirably modulate the expression or activity of one or more cellular component; or    vi) is any combination of i) to v).    
     
     
         64 . The method of  claim 59 , further comprising the step of modifying the multitargeting interfering RNA molecule, 
 i) to improve the incorporation of the first and the second strands of the multitargeting interfering RNA molecule into the RNA induced silencing complex (RISC);    ii) to increase or decrease the modulation of the expression of at least one target RNA molecule;    iii) to decrease stress or inflammatory response when the multitargeting interfering RNA molecule is administered into a subject;    iv) to alter half life in an expression system; or    iv) any combination of i) to iv).    
     
     
         65 . The method of  claim 59 , further comprising repeating the steps c) to k) of  claim 59  with a new value of n.  
     
     
         66 . The method of  claim 59 , further comprising the steps of making the designed multitargeting interfering RNA molecule and testing it in an expression system.  
     
     
         67 . The method of  claim 59 , wherein in the step of selecting a first consensus target sequence, the consensus 3′ flanking sequence to the seed comprises a sequence that is at least partially identical to the 3′ flanking sequence to the seed in at least one sequence obtained in step b) of  claim 59 .  
     
     
         68 . The method of  claim 67 , wherein the consensus 3′-flanking sequence to the seed comprises a sequence that is identical to the 3′ flanking sequence to the seed in at least one sequence obtained in step b) of  claim 59 .  
     
     
         69 . The method of  claim 59 , wherein in the step of selecting a second consensus target sequence, the consensus 3′ flanking sequence to the complete complement of the seed comprises a sequence that is at least partially identical to the 3′ flanking sequence to the complete complement of the seed in at least one sequence obtained in step b) of  claim 59 .  
     
     
         70 . The method of  claim 69 , wherein the consensus 3′ flanking sequence to the complete complement of the seed comprises a sequence that is identical to the 3′-flanking sequence to the seed in the sequences obtained in step b) of  claim 59 .  
     
     
         71 . The method of  claim 59 , wherein in the step of obtaining a first strand sequence, the complement of the consensus 3′ flanking sequence is a complete complement of the consensus 3′ flanking sequence of step h) of  claim 59 .  
     
     
         72 . The method of  claim 59 , wherein in the step of obtaining a second strand sequence, the complement of the consensus 3′ flanking sequence is a complete complement of the consensus 3′ flanking sequence of step g) of  claim 59 .  
     
     
         73 . The method of  claim 59 , wherein in the step of designing a multitargeting interfering RNA molecule, the first strand and the second strand are completely complementary to each other, excepting the overhangs if present.  
     
     
         74 . The method of  claim 59 , wherein in the step of designing a multitargeting interfering RNA molecule, the first strand and the second strand are incompletely complementary to each other.  
     
     
         75 . A method of treating a subject, comprising the step of administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a multitargeting interfering RNA molecule of  claim 1 .  
     
     
         76 . The method of  claim 75 , further comprising administering to said subject a therapeutically effective amount of one or more additional therapeutic agents.  
     
     
         77 . A method of inhibiting the onset of a disease or condition in a subject, comprising administering to said subject a prophylactically effective amount of a pharmaceutical composition comprising at least one multitargeting interfering RNA molecule of  claim 1 .  
     
     
         78 . A process for making a pharmaceutical composition comprising mixing a multitargeting interfering RNA molecule of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         79 . A method of introducing a multitargeting interfering RNA molecule comprising Formula (I) into a cell comprising the steps of: 
 i) generating a multitargeting interfering RNA molecule comprising Formula (I)      5′-p-XSY-3′ 3′-X′S′Y′-p-5′ wherein p consists of a terminal phosphate group that is independently present or absent;    wherein S consists of a first nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S′ consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S′ are at least substantially complementary to each other but are not palindromic;    wherein X, X′, Y, or Y′, is independently absent or consists of a nucleotide sequence;    wherein XSY is at least partially complementary to the first binding sequence to allow stable interaction therewith;    wherein Y′S′X′ is at least partially complementary to the second binding sequence to allow stable interaction therewith and is at least partially complementary to XSY to form a stable duplex therewith; and      ii) contacting the multitargeting interfering RNA molecule with a cell.    
     
     
         80 . The method of  claim 79  wherein the multitargeting interfering RNA is encoded by DNA.  
     
     
         81 . The method of  claim 79  wherein the RNA is encoded by a DNA or RNA vector  
     
     
         82 . The method of  claim 79  wherein the contacting step further comprises the step of introducing the RNA molecule or an RNA molecule encoded by a DNA or RNA vector into the cell.