ES2426918T3 - Modified polynucleotides to reduce non-specific effects on RNA interference - Google Patents

Abstract

A double-stranded siRNA comprising: i. a effector strand comprising a. a first 5' end effector nucleotide, wherein said first 5' end effector nucleotide comprises a first 2'-O-alkyl modification, and b. a second 5'-end effector nucleotide, wherein said second 5'-end effector nucleotide comprises a second 2'-O-alkyl modification; and ii. an antisense strand comprising a. a first 5' terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more phosphate groups attached to the 5' carbon of its sugar moiety, and b. a second 5' terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification, wherein said effector strand and said antisense strand are capable of forming an 18-19 base pair duplex of nucleotides having 100% complementarity across the range of the duplex, and within said duplex said first 5'-terminal effector nucleotide is the 5'-most nucleotide of the effector strand, said second 5'-terminal effector nucleotide is immediately adjacent and is downstream of the first 5' terminal effector nucleotide, said first 5' terminal antisense nucleotide is the 5' most nucleotide of the antisense strand and said second 5' terminal antisense nucleotide is immediately adjacent to and downstream of the first 5' terminal antisense nucleotide ', wherein all effector strand and antisense strand nucleotides other than said first 5' terminal effector nucleotide, said second nucleotide 5' terminal effector nucleotide, and said second 5' terminal antisense nucleotide comprise a 2'-OH.

Description

Modified polynucleotides to reduce non-specific effects on RNA interference

Field of the Invention

The present invention refers to the field of modified polynucleotides.

Background

It is currently believed that gene inactivation through RNA-induced gene silencing involves a minimum of three different levels of control: (i) transcription inactivation (siRNA-guided DNA and histone methylation); (ii) degradation of mRNA induced by small interference RNA (siRNA); and (iii) transcriptional attenuation induced by siRNA. The RNA interference (siRNA) generated by siRNA can have a long duration and be effective across multiple cell divisions. Therefore, RNAi represents a potentially valuable tool that can be useful in gene function analysis, drug target validation, route analysis, and disease therapy.

Recent studies on the mechanism of the RNAi-mediated transcript degradation pathway have revealed numerous key components in this route. A type II RNase called Dicer processes the long dRNA in siRNA (19-23 bp duplex) that is subsequently paired with the RNA Interference Silence Complex (RISC) to mediate the degradation of target transcripts in a specific way. sequence. This phenomenon has been observed in a diverse group of organisms. Unfortunately, initial attempts to use long mRNA to induce RNAi in mammalian cells achieved only limited success due to the induction of the interferon response, resulting in a general, as opposed to directed, inhibition of the synthesis of protein.

More recently, it has been shown that when short synthetic siRNAs are introduced into cultured mammalian cells, a specific degradation of the target mRNA sequence can be achieved without inducing an interferon response. These short duplexes can act catalytically at sub-molar concentrations to cleave more than 95% of the target mRNA in a cell. A description of the mechanisms for the activity of siRNA is provided, as well as some of its applications in Provost et al., Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, EMBOJ, 2002 Nov., 1, 21 (21): 5864 -5874; Tabara et al., The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 2002, June 28, 109 (7): 861 -71; Ketting et al., Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans, Genes and Development, 2001, 15 (20): 2654-9; and Martinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNA, Cell 2002, 6 Sept., 110 (5): 563.

Despite the promise of RNAi, four main issues, including functionality, specificity, release methods, and stability, should be addressed when working with siRNA. Specificity refers to the ability of a specific siRNA to silence a desired target without altering the expression of other genes, and recent studies have shown that inhibition of a gene other than the desired one ("off-targeting", that is, inhibition from targets other than the intended target) is much more extensive in RNAi than originally predicted (see Jackson, AL et al. (2003) "Expression profiling reveals off-target gene regulation by RNAi" Nature Biotechnology 21: 635- 7).

Since non-specific effects can induce undesirable phenotypes, new methods and compositions that minimize, alter, or eliminate non-specific effects are considered essential for siRNA to become an effective research and therapeutic tool. The present invention addresses the issue of specificity by providing modifications to the siRNA that can increase or alter the specificity of the siRNA.

Compendium of the invention

The present invention is directed to compositions and methods for performing RNA interference. In general, the chemical modifications by siRNA described herein affect a critical property of the molecules: specificity. Modifications that affect specificity are particularly advantageous in research and therapeutic applications where specificity is critical. A different combination of modifications (and derivatives of that modification pattern) is described which substantially improves the applications of RNAi and which is applicable in the design of optimal silencing reagents.

Accordingly, the invention provides: A double stranded siRNA comprising:

i. an effector strand that comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification, and

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii. an antisense strand that comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more phosphate groups attached to the 5 'carbon of its sugar radical, and

b.

 a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification,

wherein said effector strand and said antisense strand are capable of forming a duplex of 18-19 base pairs of nucleotides having a 100% complementarity along the interval of the duplex, and within said first terminal effector nucleotide 5 'is the nucleotide plus 5 'of the effector strand, said second terminal effector nucleotide 5' is immediately adjacent and is downstream of the first terminal effector nucleotide 5 ', said first terminal antisense nucleotide 5' is the nucleotide plus 5 'of the antisense strand and said second 5 'terminal antisense nucleotide is immediately adjacent and is downstream of the first 5' terminal antisense nucleotide, wherein all nucleotides of the effector strand and antisense strand other than said first 5 effector end nucleotide, said second effector nucleotide 5 'terminal, and said second 5' terminal antisense nucleotide comprise a 2'-OH.

The invention also provides a double-stranded siRNA of the invention for use in the treatment of the human or animal organism by therapy or for use in a diagnostic method that is practiced in a human or animal organism.

Brief description of the figures

The advantages of the preferred embodiments of certain aspects of the invention are shown in the attached figures, wherein: Figure 1 represents the relationship between the positioning of an embodiment of the modifications of the invention in duplexes that are longer than 25 bp. The optimal design of the molecules guarantees that after digestion with Dicer, the final functional duplex contains the modification pattern of the invention represented. Note, on the initial Dicer substrate, that the effector and / or antisense strand may have 3 'protrusions. Alternatively, both ends may have blunt ends. Figure 2 represents the relationship between the positioning of the modifications of the invention on forks. The optimal design of the molecules guarantees that after digestion with Dicer, the final functional duplex contains the modification pattern of the invention represented. Note, on the initial Dicer substrate, that the free (open) end of the molecule may be blunt or may contain a 3 'overhang. In addition, the unimolecular molecule can be organized in 5 'antisense-loop-effect or 5' effectorbucle-antisense orientation. Figure 3 illustrates an outline of the 2'-ACE RNA synthesis cycle. Figure 4 illustrates the structure of a preferred 2'-ACE protected RNA immediately before 2 'deprotection. Figures 5A and 5B represent the relationship between modification and function for siRNA SEAP-2217 2'-Omethylated. The figures demonstrate the effect on gene silencing of single base modifications (black bars) and matched (gray) 2'-O-methyl of the effector strand (Figure 5A) and antisense strand (Figure 5B) of SEAP- 2217 The X axis represents the positive relationship of the modification together with each strand of siRNA (5 '→ 3'). The Y axis represents the percentage of expression with respect to controls. Figures 6A-6F show the effects of 2'-O-methylation with and without 5 'phosphorylation on the antisense strands of six different luciferase specific siRNAs (luc 8, 18, 56, 58, 63, and 81). "S" = effector thread. "AS" = antisense thread. "*" indicates 2'-O-methylation at positions 1 and 2 of the designated strand. "p" indicates 5 'phosphorylation of the designated strand. The Y axis represents the% expression compared to control cells (not transfected). "Control" = supposedly transfected cells. Figure 7 depicts a microarray expression profile (a heat map) generated in siRNA-treated cells chosen as IGF1R-73 target in unmodified (upper part) and modified (lower part) forms. The modification pattern includes the 2'-O-methyl modification of positions 1 and 2 in both effector and antisense strands, plus carbon 5 forforylation of the ribose ring of the 5 'terminal antisense nucleotide. (IGF1R-73: 5'-UGCUGACCUCUGUUACCUC-3 ', effector) (SEQ. ID NO.1) Figures 8A and 8B represent heat maps of siRNA treated cells that they choose as target: (A) MAPK14, and (B ) MPHOSPH1. The MAPK14-193 and MPHOSH1-202 duplexes are either unmodified (upper part), modified effector strand with a 2'-O-methyl modification of positions 1 and 2 (second from above), modified antisense strand with a modification 2 '-O-methyl of positions 1 and 2 (third from above), or both strands modified with 2'-O-methyl modifications of positions 1 and 2 (bottom of each heat map). All duplexes tested contained a phosphate group on carbon 5 of the ribose ring of the 5 'terminal antisense nucleotide. The arrows indicate the position and relative levels of target silencing. (MPHOS1-202: 5'-GACAUGCGAAUGACACUAG-3 '(SEQ. ID NO. 2); Mapk14-193: 5'-CCUACAGAGAACUGCGGUU-3' (SEQ. ID NO. 3), effector thread. Figure 9 represents a summary of the number of nonspecific targets for eight different siRNAs that choose 4 separate targets MAPK14, MPHOSPH1, PTEN, and IGF1R as the target.The chemical modification pattern of the siRNAs associated with each duplex is shown at the top of each column.In addition to ( 1) without 2'-O-methyl modifications, (2) 2'-O-methyl modifications at positions 1 and 2 of the antisense strand, (3) 2'-O-methyl modifications at positions 1 and 2 of the effector strand, or (4) 2'-O-methyl modifications at positions 1 and 2 of both strands, all duplexes tested contain a phosphate group on carbon 5 of the ribose ring of the 5 'terminal antisense nucleotide. Sequences of the effector strand of the molecules in this figure include: IGF1R-73: 5 'UGCUGACCUCUGUUACCUC-3' (SEQ. ID NO. 4), Mapk14-193: 5 'CCUACAGAG AA CUGCGGUU-3 '(SEQ. ID NO. 5), Mapk14-153: 5 'GUCAUCAGCUUUGUGCCAC-3' (SEQ. ID NO. 6), MPHOS1-202: 5 'GACAUGCGAAUGACACUAG-3' (SEQ. ID NO. 7), MPHOS1-203: 5 'AGAGGAACU CUCUGCAAGC- 3 '(SEQ. ID NO. 8), PTEN 213: 5' UGGAGGGGAAUGCUCAGAA-3 '(SEQ. ID NO. 9) and PTEN 214: 5' UAAAGAUGGCACUUUCCCG-3 '(SEQ. ID NO. 10), IGF1R-73 : UGCUGACCUCUGUUACCUC-3 '(SEQ. ID NO. 11), IGFIR-71: 5' GCUCACGGUCAUUACCGAG-3 '(SEQ. ID NO. 12) Figure 10a represents a heat map showing the results of a chemical modification walk along the siRNA for MAPK14-153. The duplexes carry individual modifications or matched 2'-O-methyl modifications of various positions of the antisense strand of the siRNA combined with 2'-O-methyl modifications at positions 1 and 2 of the effector strand and a phosphate group on carbon 5 'of the ribose ring of the first antisense (terminal) nucleotide. Mapk14-153: 5 'GUCAUCAGCUUUGUGCCAC-3' (SEQ. ID NO. 13), effector thread. The letters D-R describe the following molecules:

D: not modified E 2'O-methyl modification of positions 1 and 2 of the AS strand;

F: 2'O-methyl modification of positions 1 and 2 of the AS strand without modification of the effector strand;

G: 2'O-methyl modification of positions 2 and 3 of the AS strand;

H: 2'O-methyl modification of positions 3 and 4 of the AS strand;

I: 2'O-methyl modification of positions 4 and 5 of the AS strand;

J: 2'O-methyl modification of positions 5 and 6 of the AS strand;

K: 2'O-methyl modification of positions 6 and 7 of the AS strand;

L: 2'O-methyl modification of positions 7 and 8 of the AS strand;

M: 2'O-methyl modification of positions 8 and 9 of the AS strand;

N: 2'O-methyl modification of positions 9 and 10 of the AS strand;

O: 2'O-methyl modification of positions 10 and 11 of the AS strand;

P: 2'O-methyl modification of position 1 of the AS strand;

Q: 2'O-methyl modification of position 2 of the AS strand; Y

R: 2'O-methyl modification of positions 1, 2, 11, and 12 of the AS strand

Figure 10b represents a heat map demonstrating how position 2 is a critical nucleotide in determining non-specific gene modulation. A) The redirection of siRNA to three different genes (MAPK14, KNTC2, and STK6) was examined to determine non-specific effects by microarray analysis when the duplex was not modified (top of each set of heat maps), was modified with 2'-O-methyl groups at positions 1 and 2 of the effector strand (second starting at each set of heat maps), it was modified with 2'-O-methyl groups at positions 1 and 2 of the effector strand plus 2'-O-methyl modifications at position 1 of the antisense strand (third from the top of each set of heat maps), was modified with 2'-O-methyl groups at positions 1 and 2 of the effector strand plus 2'-O-methyl modifications at position 2 of the antisense strand (fourth from the top of each set of heat maps), and was modified with 2'-O-methyl groups at positions 1 and 2 of the effector strand plus 2'-O-methyl modifications in positions 1 and 2 of the antisense strand (fifth from the top of each set of heat maps). All duplexes in this study contained a 5 'carbon phosphate group of the ribose ring of the first antisense (terminal) nucleotide. The sequences of the effector strand used in this figure include: KNTC2: 5 'GGCUUCCUUACAAGGAGAU-3' (SEQ. ID NO. 14), Mapk14-193: 5 'CCUACAGAGAACUGCGGUU-3' (SEQ. ID NO. 15), STK6: CGGGUCUUGUGUCCUUCAA-3 '(SEQ. ID NO. 16). Figure 11 compares the effects of chemical modification with mismatches of selective base pairs on non-specific effects (MAPK14-153) induced by siRNA (Mapk14-153: 5'GUCAUCAGCUUUGUGCCAC-3 '(SEQ. ID NO. 17) , effector). The eight heat maps at the top represent duplexes that are: unmodified (top), or contain 2'-O-methyl modifications at positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, or 7 and 8 of the antisense strand. All duplexes contain a 5 'carbon phosphate group of the ribose ring of the first antisense (terminal) nucleotide. The lower half of the heat map represents siRNAs that have mismatches of base pairs (between the antisense strand of the siRNA and the target molecule) incorporated into the duplex of siRNA. All duplexes contain a 5 'carbon phosphate group of the ribose ring of the first antisense (terminal) nucleotide. Figures 12a-d demonstrate a sequence-specific siRNA toxicity, independent of the target. 12a: HeLa cells were transfected with one of 90 different siRNAs that were directed to DBI (NM _020548, position 202-291). The data is presented according to the position of ARNip on the walk. The dashed line (horizontal) represents the 75% viability threshold. The framed areas indicate toxic siRNA with sequence similarity. The toxicity data (gray bars) are superimposed on the DBI mRNA expression data (black bars) for the same set of siRNA. 12b: HeLa cells were transfected with one of 48 functional siRNAs (silencing> 70%) that target 12 different genes. Data are classified based on the level of toxicity induced by siRNA. Non-toxic siRNA (gray bars), toxic siRNA (black bars). 12c: HeLa cells transfected with a subset of toxic and non-toxic siRNAs of (b) that are directed to MAPK1 (MEK1) or MAPK2 (MEK2). Toxicity data (gray bars) are presented together with mRNA expression data (black bars). The data show that there is no correlation between the level of silencing and toxicity. 12d: Dilution studies showing the effects of toxic siRNA (MAP2K2-3, SRD5A1-1, SRD5A1-3 and SRD5A2-3) in HeLa cells at varying concentrations. Toxicity values represent the average of three independent experiments (each performed in triplicate). Error bars represent the standard deviation of the mean. For the experimental protocols: 72h hours after transfection, 25 microliters of Blue Alamar dye were added to the wells containing the cells in 100 microliters of medium. The cells were then incubated (0.5 hours) at 37 ° C in a humidified atmosphere with 5% CO2. The fluorescence was subsequently measured in a Perkin Elmer WallacVector2 1420 multi-brand counter with excitation at 540 nm and emission at 590 nm. The results presented in Figure 12 are an average of nine specific data from three independent experiments carried out on different days. For the purposes of this study, the siRNAs were defined as toxic when the results of nine different experiments (taking into account typical deviations) showed that cell viability was less than 75%. For comparative gene expression levels, mRNA was quantified using Quantigene® Kits (Genospectra, Fremont, CA) for branched DNA (rDNA) analysis according to the manufacturer's instructions. The level of GAPDH mRNA (a domestic gene) was used as a reference. Figures 13a-c. sequence dependence of siRNA toxicity. HeLa cells were transfected with functional siRNAs containing 13a: AAA / UUU, 13b: GCCA / UGGC, or 13c: non-toxic motifs. The siRNA containing the motif shows a higher incidence of toxicity. The toxicity values represent the average of three independent experiments (each carried out in triplicate). Error bars represent the standard deviation of the mean. Figures 14a-1 illustrate the siRNA-mediated toxicity mediated by the RNAi pathway. 14a: Diagram of the experimental procedures used in the eIF2C2 / Ago2 expression reduction experiments. "T1" and "T2" represent "transfection 1" and transfection 2 ", respectively. The control and test siRNA were transfected at 10 nM in each transfection, 14b-14i: Control experiments demonstrating that reduced activity of the product of the eIF2C2 gene disables the RNAi path (b, d, f, and h

-

 represent the expression levels of EGFP; c, e, g, and i - show the staining by comparative Hoechst 33342). The study demonstrates that if the RNAi path is disabled with the eIF2C2 siRNA, the subsequent addition of redirect siRNA fails to silence its target. 14j: Graph showing the effects of reducing the expression of eIF2C2 on the toxicity of siRNA; 14k: Graph showing the effect of truncation of toxic siRNA by 2 nucleotides (19 units → 17 units) on the toxicity of siRNA.

141: Graph showing the effects that the chemical modifications of toxic siRNA have on the toxicity of siRNA. 14m Toxicity of 37 sequences that target luciferase in modified and unmodified forms; 14n Graph showing the level of silencing of the sequences that target luciferase in modified and unmodified forms. The toxicity values represent the average of three independent experiments (each carried out in triplicate). Error bars represent the standard deviation of the mean. Ordinary and fluorescent microscopy was used to obtain data on cell and nucleus morphology. The living cells were stained with fluorescent nuclear dye permeable to Hoechst 33342 cells (2 microg / ml, 15 minutes at 37 ° C, Molecular Probes). The photographs were taken using an InSight CCD camera of the Leica DML fluorescent microscope and the SPOT 3.5 program. Figure 15 illustrates a 5'-O-benzhydroxy-bis (trimethylsilyloxy) silyl-2'-O-bis (2-acetoxyethyl) orthoformyl-3'-O- (N, Ndiisopropyl) methyl-phosphoramidite. B is a nucleoside base such as, for example, adenosine, guanosine, cytidine, or uracil; Z is a protective group for the exocyclic amine (isobutyryl for A and G, acetyl for C. Figure 16 illustrates a 5'-O-benzhydroxy-bis (trimethylsilyloxy) -silyl-2'-O-methyl-3'-O - (N, N-diisopropyl) methyl phosphoramidite. B is a nucleoside base such as, for example, adenosine, guanosine, cytidine, or uracil; Z is a protective group for the exocyclic amine (isobutyryl for A and G, acetyl for C Figure 17 illustrates an N, N-diisopropylamino-bis (2-cyanoethyl) -phosphoramidite Figure 18 shows heat maps generated from HeLa cells treated with duplex and cyclophilin B (18a-18c) or MAP2K1 clusters (18d-18f) in unmodified and modified forms. "N" = normal, unmodified. "OTP" = modified. The results demonstrate that the addition of chemical modifications of the invention to individual duplexes generally reduces the number of nonspecific genes in 50% or more (see 18a, 18b, 18d, 18e) The combination of modifications of the invention with clusters it reduces the number of genes that are regulated downwards> 2 times above 90% (see 18c, 18f).

Detailed description

The present invention is directed to compositions and methods for carrying out RNA interference, including siRNA-induced gene silencing. Through the use of the present invention, modified polynucleotides, and derivatives thereof, can improve the efficiency of RNA interference applications.

Unless explicitly stated otherwise, or implicitly from the content, the following terms and expressions include the meanings provided below:

I rent

The term "alkyl" refers to a hydrocarbyl radical that can be saturated or unsaturated. It can comprise radicals that are linear, branched and / or cyclic.

Illustrative alkyl groups include, but are not limited to, radicals such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl. , heptadecyl, octadecyl, nonadecyl, eicosyl and alkyl groups of higher carbon numbers, as well as 2-methylpropyl, 2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl , 6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl, isopropyl, isobutyl, isopentyl, etc. The term "alkyl" also includes alkenyl groups, such as vinyl, allyl, aralkyl and alkynyl. Unless otherwise specified, alkyl groups are not substituted.

The preferred alkyl group for a 2 'modification is a methyl group with a 2' O-connection to a ribosyl radical, that is, a 2'-O-alkyl comprising a 2'-O-methyl group. A preferred 2'-O-methyl group is an unsubstituted one: -O-CH3

2'-O-alkyl modified nucleotide

The term "" 2'-O-alkyl modified nucleotide "refers to a nucleotide unit having a sugar radical, for example a deoxyribosyl radical that is modified at the 2 'position so that an oxygen atom is anchored both to the carbon atom located in the 2 'position of the sugar as an alkyl group In various embodiments, the alkyl radical consists essentially of carbons and hydrogens A particularly preferred embodiment A particularly preferred embodiment is one in which the alkyl radical is a methyl radical

Antisense thread

The term "antisense strand" as used herein refers to a polynucleotide or a region of a polynucleotide that is substantially (that is, 80% or more) or 100% complementary to a target nucleic acid of interest. An antisense strand may be comprised of a polynucleotide region that is RNA, DNA or chimeric RNA / DNA. For example, an antisense strand may be complementary, in whole or in part, to a messenger RNA molecule, a non-mRNA RNA sequence (e.g., tRNA, tRNA and mRNA)

or a DNA sequence that is either coding or non-coding. The term "antisense strand" includes the antisense region of polynucleotides that are formed from two separate strands, as well as unimolecular siRNAs that are capable of forming hairpin structures. The terms "antisense strand" and "antisense region" are intended to be equivalent and used interchangeably. The antisense strand can be modified with a diverse group of small molecules and / or conjugated products.

2 'carbon modification

The expression "modification in carbon 2 '" refers to a nucleotide unit having a sugar radical, for example a radical that is modified at the 2' position of the sugar subunit. A "2'-O-alkyl modified nucleotide" is modified in this position so that an oxygen atom is anchored to both the carbon atom located in the 2 'position of the sugar and to an alkyl group, e.g. eg, 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl, 2'-O-butyl, 2-O-isobutyl, 2'-O-ethyl -O-methyl (-OCH2CH2OCH3), and 2'-O-ethyl-OH (-OCH2CH2OH). An "effector modification in carbon 2 '" refers to a modification in the position of the 2' carbine of a nucleotide on the effector strand or within the effector region of the polynucleotide. An "antisense modification in carbon 2 '" refers to a modification in the position of carbon 2' of a nucleotide on the antisense strand or within an antisense region of the polynucleotide.

Complementary

The term "complementary" refers to the ability of polynucleotides to form base pairs with each other. Base pairs are typically formed by hydrogen bonds between nucleotide units in strands or regions of antiparallel polynucleotides. The strands or regions of complementary polynucleotides can match the bases in the Watson-Crick manner (e.g., A with T, A with U, C with G), or in any other way that allows for stable duplex formation. Complementarity is typically measured with respect to a duplex region and thereby excludes, for example, protrusions. A duplex region comprises a region of complementarity between two strands or between two regions of a single strand, for example, a unimolecular siRNA. Typically, the region of complementarity is the result of Watson-Crick base pairing.

The perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of a strand or polynucleotide region can form hydrogen bonds with each nucleotide unit of a second strand or polynucleotide region. Complementarity less than perfect refers to the situation in which some nucleotide units, but not all, of two strands or two regions can form hydrogen bonds with each other. For example, for 20 units, if only two base pairs of each strand can form hydrogen bonds with each other, the polynucleotide strands or regions show a complementarity of 10%. In the same example, if 18 base pairs of each strand or each region can form hydrogen bonds with each other, the polynucleotide strands show a 90% complementarity. Substantial complementarity refers to polynucleotide strands or regions that show a complementarity of 80% or greater.

Deoxynucleotide

The term "deoxynucleotide" refers to a nucleotide or polynucleotide that lacks an OH group in the 2 'or 3' position of a sugar radical, and / or a 2 ', 3' terminal dideoxy instead of having a hydrogen in the carbon 2 'and / or 3'.

Deoxyribonucleotide

The terms "deoxyribonucleotide" and "DNA" refer to a nucleotide or polynucleotide comprising at least one ribosyl radical having an H at the 2 'position of a ribosyl radical. Preferably a deoxyribonucleotide is a nucleotide that has an H in its 2 'position.

Downstream

A first region or nucleotide of a nucleotide strand is considered to be downstream of a second region, if the plus 5 'portion is the closest portion of that region to the 3' end of the second region (or nucleotide).

First 5 'terminal antisense nucleotide

The term "first 5 'terminal antisense nucleotide" refers to the nucleotide or region of the antisense strand that is located in the most 5' position of that strand with respect to the bases or region of the antisense strand that have the corresponding complementary bases on the strand or effector region. Thus, in an siRNA that is formed by two separate strands (that is, it is not a unimolecular or hairpin siRNA), it refers to the base plus 5 'other than the bases that are part of any 5' overhang on the antisense thread, which may be present or not. When the first 5 'terminal antisense nucleotide is part of a hairpin molecule, the term "terminal" refers to the position plus 5' with respect to the position within the terminal region and is therefore the nucleotide plus 5 'of the antisense region.

First 5 'terminal effector nucleotide

The expression "first terminal effector nucleotide 5 '" is defined in reference to the antisense nucleotide. In molecules that are formed by two separate strands (that is, it is not a unimolecular or hairpin siRNA), it refers to the nucleotide of the effector strand that is located at the 5 ′ position of the strand with respect to the bases of the effector strand that have the corresponding complementary bases on the antisense strand. Thus, in an siRNA that is formed by two separate strands (that is, it is not a unimolecular or hairpin siRNA), it is the most 5 'different base of the bases that are part of any 5' overhang on the strand or effector region, which may or may not be present. When the first 5 'terminal effector nucleotide is part of a unimolecular siRNA that is capable of forming a hairpin molecule, the term "terminal" refers to the relative position within the strand or effector region measured by the distance from the complementary base. to the first 5 'terminal antisense nucleotide.

Functional

An siRNA can be divided into five (5) groups (non-functional, semi-functional, functional, highly functional, and hyperfunctional) based on the level or degree of silencing they induce in cultured cell lines. As used herein, these definitions are based on a set of conditions in which the siRNA is transfected into said cell line at a concentration of 100 nM and the level of silencing is tested at a time approximately 24 hours later. of transfection, and not exceeding 72 hours after transfection. In this context, "non-functional siRNAs" are defined as those siRNAs that induce less than 50% (<50%) of target silencing. "Semi-functional siRNAs" induce target silencing of 50-79%. "Functional siRNAs" are molecules that induce gene silencing of 80-95%. "Highly functional siRNAs" are molecules that induce more than 95% gene silencing. "Hyperfunctional siRNAs" are a special class of molecules. For the purposes of this document, hyperfunctional siRNAs are defined as those molecules that: (1) induce more than 95% silencing of a specific target when transfected at subnanomolar concentrations (that is, less than one nanomolar); and / or (2) induce functional (or better) levels of silencing for more than 96 hours. These relative functionalities (although not intended to be absolute) can be used to compare siRNAs with a specific target for applications such as functional genomics, target identification and therapeutics.

Functional dose

A "functional dose" refers to a dose of siRNA that will be effective in causing a greater than or equal to 95% reduction in mRNA at levels of 100 nM at 24, 48, 72, and 96 hours after administration, while a "marginally functional dose" of siRNA will be effective in causing a greater or equal 50% reduction in mRNA at 100 nM within 24 hours of administration and a "non-functional dose" of RNA will cause a reduction of less than 50% at mRNA levels at 100 nM at 24 hours after administration.

Mismatch

The term "mismatch" includes a situation in which Watson-Crick base pairing does not take place between a nucleotide of an effector strand and a nucleotide of an antisense strand, where the nucleotides are flanked by a duplex comprising pairs of bases in the 5 'direction of the wrong pairing starting directly after (in the 5' direction) of the position with the wrong pairing and in the 3 'direction of the wrong pairing starting directly after (in the 3' direction) of the position with the mismatch An example of a mismatch would be an A on the other side of a G, a C on the other side of an A, a U on the other side of a C, an A on the other side of an A, a G on the other side of a G, a C on the other side of a C, and so on. It is also intended that the mismatches include a non-alkaline residue on the other side of a modified nucleotide or nucleotide, an acyclic residue on the other side of a modified nucleotide or nucleotide, a space, or an unpaired loop. In its broadest sense, an erroneous pairing as used herein includes any alteration in a given position that decreases thermodynamic stability at or in the vicinity of the position in which the alteration appears, such that thermodynamic stability of the duplex in that particular position is less than the thermodynamic stability of a pair of Watson-Crick bases in that position. Preferred mismatches include a G on the other side of an A, and an A on the other side of a C. A particularly preferred wrong match comprises an A on the other side of an A, a G on the other side of a G, a C on the other side of a C, and a U on the other side of a U.

Nucleotide

The term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide or a modified form thereof, as well as an analogue thereof. Nucleotides include species comprising purines, e.g. eg, adenine, hypoxanthine, guanine, and their derivatives and analysis, as well as pyrimidines, e.g. eg, cytosine, uracil, thymine, and their derivatives and analogs. Preferably, a "nucleotide" comprises a cytosine, uracil, thymine, adenine, or guanine radical. Preferred nucleotides, unless otherwise specified (such as, for example, when a 2 'modification, a 5' modification, a 3 'modification, a nucleobase modification, a modified internucleotide connection is specified ), include unmodified cytosine, uracil, thymine, adenine, and guanine.

Nucleotide analogs include nucleotides that have modifications in the chemical structure of the base, sugar and / or phosphate, including, but not limited to, modifications in pyrimidine at position 5, purine modifications at position 8, modifications in the exocyclic amines of cytosine, and the replacement of 5-bromouracil; and modifications in the 2'-position sugar, including but not limited to, ribonucleotides with the modified sugar in which 2'-OH is replaced by a group such as H, OR, R, halo, SH, SR, NH2 , NHR, NR2, or CN, wherein R is an alkyl radical as defined herein. It is also intended that nucleotide analogs include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2'methylribose, unnatural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.

The modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, and uracil, xanthine, inosine, and queuosine that have been modified by the substitution or addition of one or more atoms or groups. Some examples of types of modifications that may comprise nucleotides that are modified with respect to the base radicals include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, in different combinations. More specific modified bases include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N, N, dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3- methyluridine, 5-methylcytidine, 5methyluridine and other nucleotides that have a modification in the 5, 5- (2-amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3 position -methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-dezazadenosine, 6-azouridine, 6-azocytidine, 6-azotimidine 2- thiouridine, other thiobases such as 2-thouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any purine and O- and N-alkylated pyrimidine such as N6-methylladenosine, 5-methyluridine, 5-methyluridine uridino-5-oxyacetic acid, pyridine -4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as clamp G nucleotides, adenines and guanines substituted at position 8, uracils and thymine substituted at position 5, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxy alkylaminoalkyl nucleotides, and alkylcarbonylalkyl nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar radical, as well as nucleotides that have sugars or analogs thereof that are not ribosyl. For example, the sugar radicals may be, or be based on, handy, arabic, glucopyrranous, galactopyranous, 4'-thioribose, and other sugars, heterocycles, or carbocycles. It is also intended that the term nucleotide include what is known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

Additionally, the term nucleotide also includes those species that have a detectable label, such as a radioactive or fluorescent radical, or a nucleotide-anchored mass label.

Nucleotide unit

The term "nucleotide unit" refers to a single nucleotide residue and is comprised of a modified or unmodified nitrogenous base, a modified or unmodified sugar, and a modified or unmodified radical that allows the connection of two nucleotides to each other. or to a conjugate product that makes an additional connection impossible.

Non specificity

The term "non-specificity" and the term "non-specific effects" refer to any case in which an siRNA or hRNA directed against a given target causes an undesired effect by directly or indirectly interacting with another mRNA sequence, a sequence. of DNA or a cellular protein or other radical. For example, a "non-specificity effect" can occur when there is simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the effector and / or antisense strand of the siRNA or hRNA.

Outgoing

The term "outgoing" refers to one or more terminal nucleotides with unpaired bases that result from a strand or region that extends beyond the end of the complementary strand with which the first strand or region forms a duplex. One or both of the two polynucleotides or regions of polynucleotides that are capable of forming a duplex through the hydrogen bonds of the base pairs may have a 5 'and / or 3' end that extends beyond the 3 'end and / or 5 'of the complementarity shared by the two polynucleotides or regions. The single strand region that extends beyond the 3 'and / or 5' end of the duplex is referred to as a projection.

Pharmaceutically acceptable carrier

The term "pharmaceutically acceptable carrier" includes, but is not limited to, compositions that facilitate the introduction of dsRNA, dsDNA, or mRNA / rDNA hybrids into a cell and includes, but is not limited to, solvents or dispersants, coatings, agents. anti-infectives, isotonic agents, and agents that mediate the absorption time or release of the polynucleotides and siRNA of the invention.

Polynucleotide

The term "polynucleotide" refers to nucleotide polymers, and includes, but is not limited to, DNA, RNA, DNA / RNA hybrids including polynucleotide chains of deoxyribosyl radicals and regularly and irregularly alternating ribosyl radicals (that is, where the alternate nucleotide units have an -OH, then an -H, then an -OH, then an -H, and so on at the 2 'position of the sugar radical), and modifications of these classes of polynucleotides are included where the anchoring of different entities

or radicals to nucleotide units in any position. Unless otherwise specified, or made clear from the context, the term "polynucleotide" includes both unimolecular siRNA and siRNA formed by two separate strands.

Polyribonucleotide

The term "polyribonucleotide" refers to a polynucleotide comprising two or more modified or unmodified ribonucleotides and / or their analogues.

Group

The term "grouping" refers to the process whereby two or more siRNAs (preferably rationally designed siRNAs) that target a single gene are combined and introduced into a cell to induce the reduction of a gene's expression.

Ribonucleotide and Ribonucleic Acid

The term "ribonucleotide" and the term "ribonucleic acid" (RNA), refer to a modified or unmodified nucleotide or polynucleotide comprising at least one ribonucleotide unit. A ribonucleotide unit comprises an oxygen anchored to the 2 'position of a ribosyl radical having a nitrogenous base anchored in the N-glycosidic connection of the 1' position of a ribosyl radical, and a radical that allows connection to another nucleotide or It makes the connection impossible.

RNA and RNAi interference

The term "RNA interference" and the term "RNAi" are synonymous and refer to the process whereby a polynucleotide or siRNA comprising at least one ribonucleotide unit exerts an effect on a biological process. The process includes, but is not limited to, gene silencing by mRNA degradation, translation attenuation, interactions with tRNA, rRNA, rRNA, cDNA and genomic DNA, as well as methylation of DNA with accessory proteins.

Second nucleotide

The term "second nucleotide" or "nucleotide number 2" refers to the second nucleotide within a duplex region on the effector or antisense strand, counting from the 5 'end of each respective strand. The nucleotide may be paired or, for example, in the case of a mismatch, unpaired.

Second 5 'terminal antisense nucleotide

The expression "second 5 'terminal antisense nucleotide" refers to the nucleotide that is immediately adjacent to the first 5' terminal antisense nucleotide and anchored to the 3 'position of the first 5' terminal antisense nucleotide. Thus, it is the second nucleotide plus 5 'of the strand or antisense region within the set of nucleotides for which there are complementary effector nucleotides.

Second nucleotide terminal effector 5 '

The term "second terminal effector nucleotide 5 '" refers to the nucleotide that is immediately adjacent to the first terminal effector nucleotide 5' and anchored to position 3 'of the first terminal effector nucleotide 5'. Thus, it is the second nucleotide plus 5 'of the strand or effector region within the set of nucleotides for which there are corresponding antisense nucleotides.

Effector thread

The term "effector strand" refers to a polynucleotide or region that has the same nucleotide sequence, in whole or in part, as a target nucleic acid such as a messenger RNA or a DNA sequence. The term "effector strand" includes the effector region of both polynucleotides that are formed from two separate strands, as well as unimolecular siRNAs that are capable of forming hairpin structures. When a sequence is provided, by agreement, unless otherwise indicated, that is the effector strand (or region), and the presence of the complementary antisense strand (or region) is implicit. The terms "effector strand" and "effector region" are intended to be equivalent and are used interchangeably.

SiRNA or small interference RNA

The term "siRNA" and the term "small interference RNA" refer to unimolecular nucleic acids and nucleic acids that comprise two separate strands that are capable of carrying out RNA interference and that have a duplex region between 18 and 30 base pairs in length. Additionally, the term "siRNA" and the term "small interference RNA" include nucleic acids that also contain radicals other than ribonucleotide radicals, including, but not limited to, modified nucleotides, modified internucleotide connections, non-nucleotides, deoxynucleotides and nucleotide analogs above. mentioned.

The siRNAs can be duplexes and can also comprise unimolecular polynucleotides. Such unimolecular molecules comprise regions of self-complementarity (stem) by means of which the nucleotides of a polynucleotide region are paired with another region of the polynucleotide (thereby forming a duplex), and are separated by a loop. Such unimolecular molecules may vary in size and design and are referred to by a variety of names that include, but are not limited to, hairpins, small hairpin RNA (hRNA), microRNA (miRNA) and small temporary RNA (tRNA). The length of the stem region in these molecules can vary between 18 and 45 nucleotides in length. Similarly, the size of the loop may vary between 4 and 23 nucleotides and may comprise nucleotide, non-nucleotide, and nucleotide-non-nucleotide compositions.

When siRNAs are hairpins, the effector strand and the antisense strand are part of a longer molecule.

Substantial Complementarity

Substantial complementarity refers to polynucleotide strands that show a complementarity of 80% or greater.

Preferred Embodiments

According to a first embodiment, the present invention is directed to.

A double stranded siRNA comprising:

i. an effector strand that comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification, and

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effects nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii. an antisense strand that comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical, and

b.

 a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification,

wherein said effector strand and said antisense strand are capable of forming a duplex of 18-30 base pairs of nucleotides having a 100% complementarity along the interval of the duplex, and within said duplex, said first terminal effector nucleotide 5 'is the nucleotide plus 5' of the effector strand, said second terminal effector nucleotide 5 'is immediately adjacent and is downstream of the first terminal effector nucleotide 5', said first terminal antisense nucleotide 5 'is the nucleotide plus 5' of the antisense strand and said second 5 'terminal antisense nucleotide is immediately adjacent and is downstream of the first 5' terminal antisense nucleotide, wherein all the nucleotides of the effector strand and the antisense strand other than said first terminal effector nucleotide 5 ', said second 5 'terminal effector nucleotide, and said second 5' terminal antisense nucleotide comprises a 2'-OH.

The first 2'-O-alkyl modification preferably comprises 2'-O-methyl, the second 2'-O-alkyl modification preferably comprises 2'-O-methyl, and the third 2'-O-alkyl modification preferably comprises 2 ' -Omethyl.

The authors recognize that the second position of the antisense (and effector) strand of siRNAs that are between 18 and 24 bp in length, is (unlike any other base or base pair in the siRNA) a key position in mediating the silencing of targets other than the desired target (ie, nonspecific targets) and that modifications / alterations of this position can be used to eliminate the non-specific effects generated by any siRNA. As shown in Example 8 of this document, the chemical modification of position 2 effectively eliminates the non-specific effects generated by that strand measured by microarray analysis. Similarly, Example 9 demonstrates how the addition of mismatches of base pairs at position 2 (and other positions) can dramatically alter the pattern of nonspecific effects. The ability to displace or alter non-specific effects is particularly valuable in cases where downregulation of one or more nonspecific target genes induces an undesirable phenotype. The evidence that such non-specificity induced phenotypes exist and can be eliminated by alterations in position 2 of the AS strand is provided in Example 10.

The knowledge of the importance of position 2 (in the effector and / or antisense strand) makes it possible to effectively eliminate nonspecific effects through a variety of strategies. For example, the addition of a 2 'modification (such as a 2'-O-alkyl modification at this position, eg on the antisense strand, can eliminate the non-specific effects attributable to this strand. Similarly, you can substitute a base in position 2, eg, of the antisense strand, so that there is now a mismatch between the intended target mRNA and the antisense strand of the siRNA, although a wrong pairing of a single base pair (or a modification) in this position will not dramatically alter the ability of this siRNA to silence the intended target, it will alter the ability of the siRNA to silence nonspecific targets.It should be noted that erroneous pairings of base pairs at positions other than the position 2 e.g. of the antisense strand (e.g. in positions 3, 4, 5, 6, 7, or 8 of the antisense strand of an siRNA duplex in which the numbering refers to a location with with respect to end 5 of the antisense strand, position 2 being the nucleotide that is adjacent to the nucleotide plus 5 'of the antisense strand) can also reduce, eliminate, or alter specific non-specific effects. Suitable mismatches include, but are not limited to, A-G pairings, A-A pairings, G-G pairings, C-C pairings, and U-U pairings. On the other hand, the position of the mismatch is preferably in position 3, 4, 5, 6, 7, or 8 of the antisense or effector strand. Most preferably, the position of the mismatch is at position 2 of the antisense or effector strand. It should be noted that while the use of base pair substitutions such as this one can eliminate the original set of erroneous targets, new erroneous targets can be produced (resulting from the complementarity to a new set of genes). For this reason, it is more preferable that the chemical modifications described in this invention be added to the siRNA that eliminates or minimizes all non-specific effects. Thus, modifications can be used that include, but are not limited to: 1) chemical modifications of the base, sugar or internucleotide connection of nucleotide number two of the effector and / or antisense strand; (2) alterations of nucleotides or nucleotide pairs at position 2, 3, 4, 5, 6, 7, or 8 of the effector and / or antisense strand of an siRNA, including the replacement of a base or a pair of bases so as to generate a mismatch between a potential nonspecific mRNA and the effector and / or antisense strand of the siRNA; (3) alterations of nucleotides or pairs of nucleotides at position 2 of the effector and / or antisense strand of an siRNA, including the deletion of a nucleotide or a pair of nucleotides at position 2, 3, 4, 5, 6, 7, or 8 so that a bulge is generated in the non-specific transcript when it hybridizes with the effector and / or antisense strand of the siRNA; (4) alterations of nucleotides or pairs of nucleotides at position 2, 3, 4, 5, 6, 7, or 8 of the effector and / or antisense strand of an siRNA, including insertions of a nucleotide or a pair of nucleotides in position 2, so that a protuberance is generated in the effector or antisense strand of the siRNA when it hybridizes with the nonspecific mRNA message, or (5) alterations of a nucleotide or a pair of nucleotides in position 2, 3, 4, 5, 6, 7, or 8 of the effector and / or antisense strand of an siRNA, including the presence of abbasic nucleotides, or nucleotides with modifications in the C3 position of the sugar ring, to eliminate, minimize, or alter the non-specific effects in critical cases. The mismatches described above may be used in conjunction with any of the embodiments described herein to reduce non-specific effects.

That said, the siRNA of the first embodiment comprises 18-19 base pairs, excluding the protrusions. These molecules are not processed (or are poorly processed) by the Type III RNase, Dicer, and for this reason, the pattern of modifications is retained within the cell. The effector strand and the antisense strand are 100% complementary throughout the range of base pairs. Preferably, the polynucleotide is RNA.

The siRNA of the first embodiment may also contain projections of 1-6 nucleotides at any of the 5 'or 3' ends of any effector strand and / or antisense strand. However, preferably if there is any protrusion, it is at the 3 'end of the effector strand and / or the antisense strand. Additionally, preferably any of the projections is six or less bases in length, more preferably two or less bases in length. Most preferably, there are no projections, or there are projections of two bases at one or both of the effector strand and the antisense strand at the 3 'end. Because outgoing nucleotides are frequently removed by one or more intracellular enzymatic processes or events, thereby leading to a non-phosphorylated 5'nucleotide, it is preferable not to have protrusions at the 5 'end of the antisense strand. In addition, the projections may contain one or more stabilizing modifications, such as a 2'-position halogen modification or internucleotide modifications such as phosphorothioate, phosphorodithioate, or methylphosphonate modifications.

In a further reference to the first embodiment, phosphorylation of the first 5 'terminal antisense nucleotide refers to the presence of one or more 5' carbon-anchored phosphate groups of the sugar radical of the nucleotide. Preferably there is only one phosphate group.

In accordance with the present embodiment, the modification of the first and second effector nucleotides 5 'and the second antisense nucleotide 5' is a 2'-O-alkyl group. Preferably the modification is selected from the group consisting of 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl, 2'-O-butyl, 2-O-isobutyl , 2'-O-ethyl-O-methyl (-OCH2CH2OCH3), and 2'-O-ethyl-OH (-OCH2CH2OH). Most preferably, the 2'-O-alkyl modification is a 2'O-methyl radical. Additionally, there is no need for the modification to be the same in each of the first effector nucleotide 5 ', the second effector nucleotide 5', or the second antisense nucleotide. However, for practical reasons with respect to the synthesis of the molecules of the present invention, it may be desirable to use the same modification throughout the process.

Alternatively, the molecule may have an effector strand in which the effector strand comprises a first effector nucleotide 5 ', wherein said first effector nucleotide 5' comprises a first 2'-O-alkyl modification, and a second effector nucleotide 5 ', in wherein said second effector nucleotide 5 'comprises a second 2'-O-alkyl modification; and all C and U (other than any of the positions mentioned above) are modified with a 2'-O-alkyl modification; and an antisense strand, wherein said antisense strand comprises a first antisense nucleotide 5 ', wherein said first antisense nucleotide 5' is phosphorylated, and a second antisense nucleotide 5 ', wherein said second antisense nucleotide 5' comprises a third modification 2 '-O-alkyl, and all C and U (other than if it is present in the second antisense nucleotide 5') are modified with an F 2 '. In addition, these molecules may comprise a 2 nucleotide protrusion at the 3 'end of either or both strands and said protrusion may further comprise a stabilized internucleotide connection between: (1) the two nucleotides of the protrusion; and (2) the penultimate nucleotide of the projection and the final nucleotide of the duplex region, which comprises a phosphorothioate, phosphorodithioate, or methylphosphonate connection.

Alternatively, the molecule may contain an effector strand wherein the effector strand comprises a first 5 'effector nucleotide, wherein said first 5' effector nucleotide comprises 5 'deoxynucleotide; and an antisense strand, wherein said antisense strand comprises a first antisense nucleotide 5 ', wherein said first antisense nucleotide 5' is phosphorylated at carbon 5 ', and a second antisense nucleotide 5', wherein said second antisense nucleotide 5 ' It comprises a 2'-O-alkyl modification. In order for a strand to participate in gene silencing by means of the RNA interference path, the 5 'end of the strand must be phosphorylated at the 5' carbine position. The presence of a 5 'deoxynucleotide at the 5' end removes the functional group (-OH) of that strand, thereby eliminating the ability of resident kinases to add a phosphate in this position. Without a phosphate group at the 5 'end, the ability of this strand to be involved in the silencing of the correct and nonspecific target mediated by RISC is reduced.

While the invention identifies 2'-O-alkyl groups at the positions referred to above, the inventors recognize that other chemical modification groups at identical or similar positions can also be used to minimize nonspecific effects. For example, the 2 'modified nucleotide may be a 2' halogen modified nucleotide, a 2 'amine modified nucleotide, and a 2' alkyl modified nucleotide if such modifications are included under conditions that minimize non-specific effects . When the modification is a halogen, the halogen is preferably fluorine. When the 2 'modified nucleotide is a 2' modified amine nucleotide, the amine is preferably -NH2. When the 2'-modified nucleotide is a 2'-alkyl modification, preferably the modification is selected from the group consisting of a methyl, ethyl, propyl, isopropyl, butyl, or isobutyl radical. Most preferably, a 2 'methyl modification is used, wherein the carbon of the methyl radical is directly anchored to the 2' carbon of the sugar radical.

As stated above, the modification pattern of the first embodiment is applicable to molecules that are not processed by Dicer. Although they are not part of the presently claimed invention, longer duplexes (eg, 25-30 base pairs) may also be useful. These molecules are potential substrates for Dicer and for that reason, displacements in the modification pattern and different patterns of length and position of the projections must be incorporated into the duplex design to ensure that the entire complement of modifications described in the first embodiment is present in the final duplex, after processing by Dicer. In a non-limiting example, the structure of the duplex ends is designed to ensure that the final product carries the necessary modifications in the appropriate positions. The side on which Dicer enters a duplex may be influenced by the presence or absence of projections. In addition, the position of the Dicer cleavage can be manipulated by varying the length of the projection 3 '. Thus, for example, a preferred design for molecules that have 26 base pairs or more includes: (1) a 1, 2, or 3 nucleotide projection at the 3 'end of the effector strand, and no projection in the opposite end of the duplex; (2) a 2'-O-alkyl modification in the second antisense nucleotide (counting from the 5 'end of the strand); (3) 5 'carbon phosphorylation of the first antisense nucleotide; and (4) 2'-O-alkyl modifications matched at nucleotides 21 and 12, 22 and 23, or 23 and 24 of the effector strand (counting from the 3 'end of the effector strand, projection included). The addition of the 3 'effector strand influences the side where Dicer enters the duplex (that is, Dicer will preferably enter the side of the molecule containing the protrusion rather than the blunt end of the molecule). This guarantees the preservation / retention of both antisense modifications (that is, the 2'-O-alkyl modification in the second antisense nucleotide, and the 5 'phosphate group in the first antisense nucleotide) in the final siRNA, after processing with Dicer . In addition, the positions of the m modifications of the effector strand ensure that after processing with Dicer, these modifications will be present in the terminal effector nucleotides (effector nucleotides 1 and 2) of the final, processed siRNA (see Figure 1).

According to a second embodiment, the present invention refers to a unimolecular siRNA capable of forming a hairpin siRNA, said unimolecular siRNA comprising:

to.

 an effector strand, which comprises

i. a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification, and

ii. a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

b.

 an antisense strand, which comprises

i. a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical, and

ii. a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification, and

a loop region, wherein said loop region is located between said effector strand and said antisense strand and wherein said effector strand and said antisense strand are capable of forming a duplex of 18-19 base pairs of nucleotides having a complementarity of 100 % over the interval of the duplex, and within said duplex said first 5 'terminal effector nucleotide is the nucleotide plus 5' of the effector strand, said second 5 effector terminal nucleotide is immediately adjacent and is downstream of the first effector nucleotide 5 'terminal, said first 5' terminal antisense nucleotide is the most 5 'nucleotide of the antisense strand and said second 5' terminal antisense nucleotide is immediately adjacent and is downstream of the first 5 'terminal antisense nucleotide, where all the nucleotides of the effector strand and the antisense strand other than said first 5 'terminal effector nucleotide, said second effector nucleotide 5 'terminal, and said second 5' terminal antisense nucleotide comprise a 2'-OH.

According to this embodiment, the range of modifications is the same as that of the first embodiment. However, because the polynucleotide is unimolecular and is capable of forming a hairpin, and not two separate strands, there is a contiguous molecule that comprises both an effector region and an antisense region. The effector region and the antisense region are 100% complementary. Preferably, the full length of the unimolecular siRNA contains less than 100 bases, more preferably less than 85 bases. Preferably the nucleotide is RNA.

The forks comprise two main components including a stem (which is a double-strand region that matches the antisense strand and the effector strand) and a loop. Optionally, a protruding sequence can be added to the 5 ', 3' end or both of the molecule. Preferably, when a projection is present, it is associated with the 3 'end of the molecule. When designing a unimolecular siRNA, the hairpin can be designed as a fork to the left (e.g., 5'-AS-Loop-S) or a fork to the right (e.g., 5'-SBucle- ACE). Preferably, the fork is to the left. This construction is desirable because it is easier to phosphorylate the terminal antisense nucleotide. As with the double stranded siRNA, the stem length of the molecule determines whether or not it will be processed by Dicer. Since the forks containing longer loop structures are substrates for Dicer, the position of the chemical modifications must be adjusted to ensure that the final post-Dicer processing product contains the modifications at the desired positions. Preferably, the post-Dicer processed molecule contains all the modifications described in the first embodiment. In a non-limiting example, preferred fork designs for unimolecular molecules that have stems that are longer than 24 bases include the following properties: (1) a fork design to the left, (2) a 3 'projection of 1 -3 nucleotides, (3) a 5 'carbon phosphate group of the nucleotide plus 5', (4) a 2'-O-alkyl (preferably an O-methyl) modification of the second antisense nucleotide, and (5) modifications 2 '-O-alkyl matched at nucleotides 21 and 22, 22 and 23, or 23 and 24 of the effector strand (counting from the 3' end of the effector strand, projection included). The addition of the projection of the effector strand 3 'intensifies Dicer's ability to enter the fork at the AS 5' end of the molecule. In addition, the positions of the effector strand modifications ensure that after processing with Dicer, these modifications will be present at the effector terminals of the nucleotide (effector nucleotides 1 and 2 that have complementary bases in the antisense strand) of the final processed siRNA (see Figure 2).

The fork may comprise a loop structure, which preferably comprises four to ten bases. The bases of the loop can be modified. Alternatively, the loop may comprise non-nucleotide components such as those described in U.S. Patent Application No. 10/635108, published March 25, 2004, as US 2004/0058886 A1. Preferable sequences of the loop structure include, for example, 5'-UUCG-3 '(SEQ. ID NO. 18), 5'-UUUGUGUAG-3' (SEQ. ID NO. 19), 5'-CUUCCUGUCA- 3 '(SEQ. ID NO. 20), 5'-AUAUGUG-3' (SEQ. ID NO. 21), or any other previously identified loop or pre-miRNA.

As previously described, the unimolecular siRNA of the present invention can be ultimately processed by cellular machinery so that it becomes two separate strands. Additionally, these unimolecular siRNAs can be introduced into the cell without including all modifications, and modified in the cell itself through the use of natural processes or processing molecules that have been introduced (eg, phosphorylation in the cell). However, preferably the siRNA is introduced with all the modifications already present. (Similarly, the strands of the first embodiment are preferably introduced into the cell with all modifications, although the antisense strand could, for example, be modified after introduction). If a fork is processed by Dicer, the resulting fork retains the modifications of the different embodiments described herein.

The modifications described above should not be considered to suggest that no other radical can be modified in addition to the nucleotides described, which also contribute to minimizing non-specific effects or intensifying other properties of siRNA (such as better stability or functionality). Other types of modifications are possible as long as they do not unacceptably increase non-specific effects. In certain embodiments, such additional modifications may be added to one, two, three, or more consecutive nucleotides.

or every two nucleotides of the effector strand. Alternatively, additional modifications can be confined to specific positions that have been identified as being key to access to the effector strand and / or use by RISC. As previously mentioned, further, additional modifications, such as 2'-O-alkyl groups (or other 2 'modifications) can be added to one or more, preferably all, pyrimidines (eg, nucleotides C and / or U) of the effector strand and / or 2'-F modifications (or modifications with other halogens) can be added to one or more, preferably all pyrimidines (e.g., nucleotides C and / or U) of the antisense strand other than nucleotides that are otherwise modified as specified above. Modifications such as 2'-F or 2'-O-alkyl of some or all of the C and U of the antisense and / or effector strand (respectively) and other nucleotides specified above can greatly enhance the stability of the siRNA / HRNA carrying the modifications described in embodiments 1 and 2, without appreciably altering the specific silencing of the target.

Additionally, if a label is used in conjunction with the invention, these agents may be useful as tracking agents, which could aid in the detection of transfection, as well as in the detection of where in the cell the molecule is present. Examples of commonly used marks include, but are not limited to, a fluorescent mark, a radioactive mark or a mass mark.

Additionally, stabilization modifications that are directed to the phosphate main chain can also be included in the siRNAs for some applications of the present invention. For example, at least one phosphorothioate and / or methylphosphonate may substitute for the phosphate group at some or all 3 'positions of any or all pyrimidines of the effector and / or antisense strands of the oligonucleotide backbone chain, as well as any projection. , loop structure or stem structure that may be present. Phosphorothioate (and methylphosphonate) analogs arise from the modification of phosphate groups in the oligonucleotide backbone. In phosphorothioate, the phosphate O-is replaced by a sulfur atom. In methylphosphonates, oxygen is substituted by a methyl group. Additionally, 3 'modifications of phosphorothioate can be used instead of, and independently of 2'-fluoro modifications to increase the stability of an siRNA molecule. These modifications may be used in combination with the other modifications described herein, or regardless of those modifications in the applications of siRNAs.

In other embodiments, there may be even more modifications such as those described below: an antisense strand containing a 2'-O-alkyl modification in the second antisense nucleotide, plus a phosphate group on carbon 5 of the first antisense nucleotide; plus the first effector nucleotide counting from the 5 'end of that strand is a 5' deoxynucleotide. This can be beneficial by substantially reducing the non-specific effects induced in both the effector and antisense strands.

In another embodiment, the molecule comprises the following modifications:

(one)

 the second terminal antisense nucleotide contains a 2'-O-alkyl modification; Y

(2)

 the first terminal antisense nucleotide contains a 5 'carbon phosphate group; Y

(3)

 The 5 'carbon of the first terminal nucleotide of the effector strand comprises any group known in the art that is suitable for blocking preventing the hydroxyl group from accepting or converting into a phosphate group. Preferably, the group that blocks the 5'-terminal nucleotide comprises a 5'-O-alkyl group, a group that blocks an amine 5, or a group that blocks a 5 'azide.

In another embodiment, any of the compositions of the present invention may additionally comprise a 3 'terminal protection. The 3 'terminal protection can be, for example, an inverted deoxythymidine.

In other embodiments of the present invention, any of the compositions may comprise a conjugate product. The conjugate product can be selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof. The conjugate product may be, for example, cholesterol or PEG. The conjugate product may additionally comprise a label, such as, for example, a fluorescent label. The fluorescent brand can be selected from the group consisting of TAMRA, BODIPY, Cy3, Cy5, fluorescein, and Dabsilo. Alternatively, the fluorescent brand can be any fluorescent brand known in the art.

The above-described modifications of the present invention can be combined with an siRNA containing sequences that were randomly selected, or in accordance with any rational design selection procedure, for example, the rational design algorithm described in the Patent Application the United States with Serial No. 10 / 714,333, filed on November 14, 2003, entitled "Functional and Hyperfunctional siRNA"; in International Patent Application number PCT / US2003 / 036787, published on June 3, 2004 as WO 2004/045543 A2, entitled "Functional and Hyperfunctional siRNA; and in the United States Patent Application with Serial No. 10/940892, filed on September 14, 2004, entitled "Methods and Compositions for Selecting siRNA of Improved Functionality." Additionally, it may be desirable to select sequences based entirely or in part on internal thermal stability, which may facilitate Processing by cellular machinery.

It should be noted that the modifications of the first and second embodiments of the present invention may have different effects depending on the functionality of the siRNAs used. Thus, in highly functional siRNAs, the modifications of the present invention can cause a molecule to lose a certain amount of functionality, but would nevertheless be desirable since non-specific effects are reduced. In contrast, when moderately or poorly functional siRNAs are used, there is a very small decrease in functionality and in some cases, the functionality may increase.

Several approaches can be used to identify both the type of molecule and the key position necessary to eliminate non-specific effects on the effector and / or antisense strand. In a non-limiting example, a function modification walk was made. In this procedure, a single type of modification is added to one or more nucleotides along the effector and / or antisense strand. Subsequently, the modified and unmodified molecules are tested for: (1) functionality; and (2) non-specific effects, by one of several methods. Thus, for example, groups 2'-O-Me can be added in positions 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, or 18 and 19 of the effector and / or antisense strand and tested for functionality (e.g., measuring the ability of these molecules to silence specific targets) and non-specific effects. If key positions are identified that eliminate some or all of the non-specificities, but which result in a loss of functionality of the duplex, a second round of modification walks is made, through which additional chemical groups can be added (e.g. ., 5 'phosphate at the 5' end of the antisense strand), mismatches, or bumps that are suspected to increase the functionality of the duplex, to molecules that already contain the modification that eliminates non-specificity.

In order to determine which modifications are permissible, several non-limiting analyzes can be carried out to identify the modifications that limit the non-specific effects. In a non-limiting example, the effector or antisense strand (which carries the modification being tested) can be labeled with one of the many labeled nucleotides. Subsequently, an analysis can be carried out by means of which the affinity of RISC can be compared, e.g. eg, by the effector strand modified with that of the unmodified form. Alternatively, siRNAs containing various modifications can be transfected into cells by a variety of methodologies and cultures can be subsequently evaluated by microarray analysis to determine if the modifications alter the number and pattern of genes other than the target gene. .

According to a third embodiment, the present invention is directed to a method for minimizing non-specific effects, said method comprising exposing a modified siRNA containing the modifications described in embodiment 1, to a target nucleic acid, or to a cell or organism which either expresses the target nucleic acid or is capable of expressing the target nucleic acid.

According to another embodiment, the present invention is directed to a method for minimizing non-specific effects, said method comprising exposing a unimolecular siRNA containing the modifications described in the second embodiment to a target nucleic acid, or to a cell or organism that or It either expresses the target nucleic acid or is capable of expressing the target nucleic acid.

In various embodiments, a 3 'overhang of 1 to 6 bases may be present in at least one of the effector strand and the antisense strand. The projection may be present with any of the embodiments described herein herein, unless otherwise specified or implied in the context.

The siRNAs of the present invention can be used to prepare a kit, comprising at least two siRNAs, wherein at least two siRNAs comprise a first siRNA and a second siRNA, and wherein each of the first siRNA and the second siRNA comprises:

i. to the effector strand, wherein said effector strand comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification; Y

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii. an antisense strand, wherein said antisense strand comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical; Y

b.

 a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification;

wherein said effector strand and said antisense strand are capable of forming a duplex of 18-19 base pairs of nucleotides, wherein the duplex has a 100% complementarity along the interval of the duplex, and within said duplex said first 5 'terminal effector nucleotide is the most 5' nucleotide of the effector strand, and said second terminal effector nucleotide 5 'is immediately adjacent and is downstream of the first terminal effector nucleotide 5'; said first 5 'terminal antisense nucleotide is the most 5' nucleotide of the antisense strand and said second 5 'terminal antisense nucleotide is immediately adjacent and is downstream of the first 5' terminal antisense nucleotide, where all nucleotides of each strand other than said first 5 'terminal effector nucleotide, said second 5' terminal effector nucleotide, and said second 5 'terminal antisense nucleotide comprises a 2'-OH.

Such a kit may comprise siRNAs wherein the first siRNA and the second siRNA each comprise a sequence that is at least substantially complementary to a region in the target mRNA. The kit may comprise an siRNA wherein the region of the target mRNA at which the sequence of the first siRNA is at least substantially complementary to the region of the target mRNA to which the sequence of the second siRNA is at least substantially complementary. The kit may comprise siRNA so that the region of the target mRNA at which the sequence of the first siRNA is at least substantially complementary does not overlap with the region of the target mRNA at which the sequence of the second siRNA is at least substantially complementary.

The siRNAs of the second embodiment can be used to prepare a kit, comprising at least two unimolecular siRNAs, wherein at least two siRNAs comprise a first siRNA and a second siRNA, and wherein each of the first siRNA and the second siRNA comprises :

i. an effector region, wherein said effector region comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification; Y

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii. an antisense region, wherein said antisense region comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical; Y

b.

 a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification;

wherein said effector region and said antisense region are capable of forming a duplex of 18-19 base pairs of nucleotides, wherein the duplex has a 100% complementarity along the interval of the duplex, and within said duplex said first 5 'terminal effector nucleotide is the most 5' nucleotide of the effector region, and said second terminal effector nucleotide 5 'is immediately adjacent and is downstream of the first terminal effector nucleotide 5'; said first 5 'terminal antisense nucleotide is the most 5' nucleotide of the antisense region and said second 5 'terminal antisense nucleotide is immediately adjacent and is downstream of the first 5' terminal antisense nucleotide, where all the nucleotides of the effector region and the antisense region other than said first 5 'terminal effector nucleotide, said second 5' terminal effector nucleotide, and said second 5 'terminal antisense nucleotide comprise a 2'-OH.

The kit may comprise siRNA so that the first siRNA and the second siRNA each comprise a sequence of at least substantially complementary to a region in a target mRNA. In some embodiments, the kit comprises siRNA so that the region of the target RNA to which the sequence of the first siRNA is at least substantially complementary overlaps with the region of the target mRNA to which the sequence of the second siRNA is at least substantially complementary. . In some embodiments, the kit comprises siRNA so that the region of the target mRNA at which the sequence of the first mRNA is at least substantially complementary does not overlap with the region of the target mRNA at which the sequence of the second is at least substantially complementary. SiRNA

The siRNAs of the second embodiment can be used to carry out a method to minimize non-specific effects in ex vivo RNAi, said method comprising exposing at least two unimolecular siRNAs to a target nucleic acid or to a cell, wherein at least two unimolecular siRNAs comprise a first unimolecular siRNA and a second unimolecular siRNA, wherein each of the first unimolecular siRNA and the second unimolecular siRNA comprises

i. an effector region, wherein said effector region comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification; Y

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii. an antisense region, wherein said antisense region comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical; Y

b.

 a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification;

wherein said effector region and said antisense region are capable of forming a duplex of 18-19 base pairs of nucleotides, wherein the duplex has a 100% complementarity along the duplex interval, and within said duplex said first terminal effector nucleotide 5 'is the nucleotide plus 5' of the effector region, and said second terminal effector nucleotide 5 'is immediately adjacent and is downstream of the first terminal effector nucleotide 5'; said first 5 'terminal antisense nucleotide is the most 5' nucleotide of the antisense region and said second 5 'terminal antisense nucleotide is immediately adjacent and is downstream of the first 5' terminal antisense nucleotide, where all the nucleotides of the effector region and the antisense region other than said first 5 'terminal effector nucleotide, said second 5' terminal effector nucleotide, and said second 5 'terminal antisense nucleotide comprise a 2'-OH.

The method may comprise the use of siRNA wherein the first unimolecular siRNA and the second unimolecular siRNA each comprise a sequence that is at least substantially complementary to a region of a target mRNA. The method may comprise the use of siRNA so that the region of the target mRNA at which the first unimolecular mRNA sequence is at least substantially complementary overlaps with the region of the target mRNA at which the second second sequence is at least substantially complementary. Unimolecular siRNA. The method may comprise the use of siRNA so that the region of the target mRNA at which the sequence of the first unimolecular mRNA is at least substantially complementary does not overlap with the region of the target mRNA at which the sequence of the at least substantially complementary second unimolecular siRNA.

The siRNAs of the second embodiment can be used to carry out a method to minimize non-specific effects on interference with ex vivo RNA, said method comprising exposing at least two siRNAs to a target nucleic acid or to a cell, where the al at least two siRNAs comprise a first siRNA and a second siRNA, wherein each of the first siRNA and the second siRNA comprises

i. an effector strand, wherein said effector strand comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification; Y

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii. an antisense strand, wherein said antisense strand comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical; Y

b.

 a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification;

wherein said effector strand and said antisense strand are capable of forming a duplex of 18-19 base pairs of nucleotides, wherein the duplex has a 100% complementarity along the interval of the duplex, and within the duplex said first nucleotide terminal effector 5 'is the nucleotide plus 5' of the effector strand, and said second terminal effector nucleotide 5 'is immediately adjacent and is downstream of the first terminal effector nucleotide 5'; said first 5 'terminal antisense nucleotide is the most 5' nucleotide of the antisense strand and said second 5 'terminal antisense nucleotide is immediately adjacent and is downstream of the first 5' terminal antisense nucleotide, where all nucleotides of each strand other than said first 5 'terminal effector nucleotide, said second 5' terminal effector nucleotide, and said second 5 'terminal antisense nucleotide comprise a 2'-OH.

The method may comprise the use of siRNA so that the first siRNA and the second siRNA each comprise a sequence that is at least substantially complementary to a region of a target mRNA. The method may comprise the use of siRNA so that the region of the target mRNA at which the sequence of the first siRNA is at least substantially complementary overlaps with the region of the target mRNA at which the sequence of the second siRNA is at least substantially complementary. . The method may comprise the use of siRNA so that the region of the target mRNA at which the sequence of the first mRNA is at least substantially complementary does not overlap with the region of the target mRNA at which the sequence of the second is at least substantially complementary. SiRNA

Because the ability of the mRNA of the present invention to retain functionality and show improved specificity does not depend on the sequence of the bases, the type of cell, or the species into which it is introduced, the present invention is applicable to a wide range of organisms, including, but not limited to, plants, animals, protozoa, bacteria, viruses and fungi. The present invention is particularly advantageous for use in mammals such as cattle, horses, goats, pigs, sheep, canids, rodents such as hamsters, mice, and rats, and primates such as gorillas, chimpanzees, and humans.

The present invention can be advantageously used with various cell types, including, but not limited to, primary cells, germ cell lines and somatic cells. The cells can be, for example, pluripotential cells or differentiated cells. For example, cell types can be embryonic cells, oocytes, spermatozoa, adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes , granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and endocrine or exocrine gland cells.

The present invention is applicable for use in interference with RNA (and / or use as a control) directed against a wide range of genes, including but not limited to, the 45,000 genes of the human genome, such as those involved in diseases such as diabetes, Alzheimer's and cancer, as well as genes of the genomes of humans, mice, hamsters, chimpanzees, goats, sheep, horses, camels, pigs, dogs, cats, nematodes (e.g., C. elegans) , flies (e.g., D. melanogaster), and other vertebrates and invertebrates.

The siRNAs of the present invention can be administered to a cell by any method that is currently known or will be known and that which, from reading this description, one skilled in the art would conclude that it is useful with the present invention For example, siRNAs can be passively released into cells. Passive absorption of modified siRNAs can be modulated, for example, by the presence of a conjugate product such as a polyethylene glycol radical or a cholesterol radical at the 5 'end of the effector strand and / or, in appropriate circumstances, a pharmaceutically acceptable carrier.

Other methods for release include, but are not limited to, transfection techniques that employ DEAE-Dextran, calcium phosphate, cationic lipids / liposomes, microinjection, electroporation, immunoporation, and coupling of siRNAs to specific conjugated products or ligands such as antibodies, peptides, antigens, or receptors.

Preferably, siRNAs comprise duplex when administered.

Additionally, the method of assessing the level of gene silencing is not limited. Thus, the silencing capacity of any given siRNA can be studied by any of the numerous procedures tested in the art including, but not limited to Northern analysis, Western analysis, RT PCR, expression profiling, and others.

The polynucleotides of the present invention can be synthesized by any method that is currently known or that is to be known and that from reading this description, one skilled in the art would conclude that it is useful for synthesizing the molecules of the present invention. The siRNA duplexes containing the specified modifications can be chemically synthesized using compositions of materials and methods described in Scaringe, S.A. (2000) "Advanced 5'-silyl-2'-orthoester approach to RNA oligonucleotide synthesis", Methods Enzymol. 317, 3-18; Scalinge, S.A. (2001) "RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry," Methods 23, 206-217; Scaringe, S. and Caruthers, M.H. (1999) United States Patent No. 5,889,136; Scaringe, S. and Caruthers, M.H. (1999) United States Patent No. 6,008,400; Scaringe, S. (2000) United States Patent No. 6,111,086; Scaringe, S. (2003) United States Patent No.

6,590,093. The synthesis method uses 5'-O-silyl-2'-O-orthoester-3'-O-phosphoramidites with the protected nucleoside base to assemble the desired unmodified siRNA sequence on a solid support in the 3 'to 5 direction '. In summary, the synthesis of the required phosphoramidites begins from the conventional protected base ribonucleosides (uridine, N4-acetylcytidine, N2-isobutyrylguanosine and N6-isobutyryladenosine). The introduction of the 5'-O-silyl and 2'-O-orthoester protecting groups, as well as the reactive 3'-O-phosphoramidite radical is then completed in five steps, including:

one.

 Simultaneous transient blocking of the 5'- and 3'-hydroxyl groups of the nucleoside sugar with Markiewicz reagent (1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane [TIPS-Cl2]) in pyridine solution {Markiewicz , WT (1979) "Tetraisopropyldisiloxane-1,3-diyl, a Group for Simultaneous Protection of 3'- and 5'-Hydroxy Functions of Nucleosides", J. Chem. Research (S), 24-25}, followed by chromatographic purification;

2.

 Regiospecific conversion of TIPS-nucleoside 2'-hydroxyl sugar in bis (acetoxyethyl) orthoester [ACE derivative] using tris (acetoxyethyl) orthoformate in dichloromethane with pyridinium p-toluenesulfonate as catalyst, followed by chromatographic purification;

3.

 Release of the 5'- and 3'-hydroxyl groups from the nucleoside sugar by specific release of the TIPS protecting group using hydrogen fluoride and N, N, N "N'-tetramethylethylenediamine in acetonitrile, followed by chromatographic purification;

Four.

 Protection of 5'-hydroxyl in the form of a 5'-O-silyl ether using benzhydroxybis (trimethylsilyloxy) silyl chloride [BzH-Cl] in dichloromethane, followed by purification; Y

5.

 Conversion into the 3'-O-phosphoramidite derivative using bis (N, N-diisopropylamino) methoxyphosphine and 5-methylthio-1H-tetrazol in dichloromethane / acetonitrile, followed by chromatographic purification.

Phosphoramidite derivatives are typically thick syrups, colorless to pale yellow. For compatibility with automatic RNA synthesis instruments, each of the products is dissolved in a predetermined volume of anhydrous acetonitrile, and this solution is divided into aliquots in the appropriate number of serum vials to provide an amount of 1.0 mmol of phosphoramidite in each vial. The vials are then placed in a suitable vacuum dryer and the solvent is removed under high vacuum overnight. The atmosphere is then replaced by dry argon, the vials are covered with rubber diaphragms, and the packed phosphoramidites are stored at -20 ° C until necessary. Each phosphoramidite is dissolved in anhydrous acetonitrile sufficient to give the desired concentration before installation in the synthesis apparatus.

The synthesis of the desired oligoribonucleotide is carried out using an automatic synthesis instrument. This begins with the 3'-terminal nucleoside covalently linked through its 3 'hydroxyl to a solid-shaped polystyrene support by means of a cleavable connection. The appropriate amount of support for the desired synthesis scale is measured in a reaction cartridge, which is then fixed to the synthesis apparatus. The bound nucleoside is protected with a 5'-O-dimethoxytrityl radical, which is removed with anhydrous acid (dichloroacetic acid in 3% dichloromethane [v / v]) in order to release 5'-hydroxyl for assembly of the chain.

Subsequently, the nucleosides of the sequence to be assembled are successively added to the growing chain on the solid support using a four-stage cycle, consisting of the following general reactions:

one.

Coupling: the appropriate phosphoramidite is activated with 5-ethylthio-1H-tetrazole and allowed to react with the free 5'-hydroxyl of the nucleoside or oligonucleotide attached to the support. Optimization of the concentrations and molar excesses of these two reagents, as well as the reaction time, results in coupling yields generally above 98% per cycle.

2.

 Oxidation: the internucleotide connection formed in the coupling stage leaves the phosphorus atom in its oxidation state P (III) [phosphite]. The biologically relevant oxidation state is P (V) [phosphate]. The phosphorus is therefore oxidized from P (III) to P (V) using a test solution of butylhydroperoxide in toluene.

3.

 Terminal protection: the small amount of 5'-hydroxyl groups that have not reacted should be blocked against participation in subsequent coupling cycles in order to avoid the formation of sequences containing deletions. This is achieved by treating the support with a large excess of acetic anhydride and 1-methylimidazole in acetonitrile, which effectively blocks the residual 5'-hydroxyl group in the form of acetate esters.

Four.

 De-silylation: silyl-protected 5'-hydroxyl must be unprotected before the next coupling reaction. This is achieved through treatment with triethylamine - hydrogen trifluoride in N, N-dimethylformamide, which quickly and efficiently releases 5'-hydroxyl without the concomitant removal of other protective groups (2'-O-ACE, N-base protecting groups -acylated, or phosphate methyl).

It should be noted that among the four previous reaction steps there are several washes with acetonitrile, which are used to remove excess reagents and solvents before the next reaction stage. The previous cycle is repeated the necessary number of times until the unmodified portion of the oligoribonucleotide has been assembled. The above synthesis method is illustrative only and should not be considered as limiting the means by which the molecules can be made. Any method that is currently known or that will be known to synthesize siRNA can be used and that, upon reading this description, one skilled in the art would conclude that it is useful in relation to the present invention.

SiRNA duplexes of certain embodiments include two modified nucleosides (e.g., 2'-O-methyl derivatives) at the 5 'end of each strand. The 5'-O-silyl-2'-O-methyl-3'-O-phosphoramidite derivatives required for the introduction of these modified nucleosides are prepared using procedures similar to those previously described (steps 4 and 5 above), starting from 2'-O-methyl nucleosides with the protected base (2'-O-methyl-uridine, 2'-Omethyl-N4-acetylcytidine, 2'-O-methyl-N2-isobutyrylguanosine and 2'-O-methyl-N6 -isobutyryladenosine). The absence of 2'hydroxyl in these modified nucleosides eliminates the need for ACE protection of these compounds. As such, the introduction of 5'-O-silyl and the reactive 3'-O-phosphoramidite radical is completed in two steps, including:

one.

 Protection of 5'-hydroxyl in the form of a 5'-O-silyl ether using benzhydroxybis (trimethylsilyloxy) silyl chloride (BzH-Cl) in N, N-dimethylformamide, followed by chromatographic purification; Y

2.

 Conversion into the 3'-O-phosphoramidite derivative using bis (N, N-diisopropylamino) methoxyphosphine and 5-methylthio-1H-tetrazole in dichloromethane / acetonitrile, followed by chromatographic purification.

The packaging of phosphoramidites after purification is carried out using the procedures described above for conventional nucleoside phosphoramidites. Similarly, the incorporation of the two 5'-O-silyl-2'-O-methyl-nucleosides through their phosphoramidite derivatives is completed by applying twice the same four-stage cycle described above for conventional nucleoside-phosphoramidites. .

The siRNA duplexes of certain embodiments of this invention include a phosphate radical at the 5 'end of the antisense strand. This phosphate is chemically introduced as the final coupling to the antisense sequence. The required phosphoramidite derivative (bis (cyanoethyl) -N, N-diisopropylamino-phosphoramidite) is synthesized as follows: phosphorus trichloride is treated with an equivalent of N, N-diisopropylamine in anhydrous tetrahydrofuran in the presence of excess triethylamine . Next, two equivalents of 3-hydroxypropionitrile are added and allowed to react completely. Finally, the product is purified by chromatography. Post-purification phosphoramidite packaging is carried out using the procedures described above for conventional nucleoside phosphoramidites. Similarly, the incorporation of phosphoramidite at the 5 'end of the antisense strand is completed by applying the same four-stage cycle previously described for conventional nucleoside phosphoramidites.

The modified protected oligonucleotide remains connected to the solid support at the end of the chain assembly. A two-stage rapid cleavage / deprotection method is used to remove the methyl phosphate protecting groups, cleaving the solid support oligoribonucleotide, and removing the N-acylated base protecting groups. It should be noted that this procedure also removes the cyanoethyl 5'phosphate protecting groups from the antisense strand. Additionally, the process eliminates the acetyl functionalities of the ACE orthoester, converting the 2'-O-ACE protecting group into the bis (2-hydroxyethyl) orthoester. This new orthoester is significantly more labile to weak acids as well as more hydrophilic than the parental ACE group. The two-stage procedure is summarized below:

one.

 The oligoribonucleotide bound to the support is treated with a solution of disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in N, N-dimethylformamide. This reagent quickly and efficiently removes methyl protecting groups from internucleotide phosphate connections without cleaving the solid support oligoribonucleotide. The support is then washed with water to remove excess dithiolate.

2.

 The oligoribonucleotide is cleaved from the solid support with 40% aqueous methylamine (w / v) at room temperature. The methylamine solution containing the crude oligoribonucleotide is heated to 55 ° C to remove the protective groups from the nucleoside bases. The crude orthoester protected oligoribonucleotide is obtained after removal of the solvent in vacuo.

The elimination of 2'-orthoesters is the final stage in the synthesis procedure. This is completed by treating the crude oligoribonucleotide with an aqueous solution of acetic acid and N, N, N ', N'-tetramethylethylenediamine, pH 3.8, at 55 ° C for 35 minutes. The completely unprotected oligoribonucleotide is then subjected to removal of the salts by ethanol precipitation and is isolated by centrifugation.

In addition, the incorporation of fluorescent labels at the 5 'end of a polynucleotide is a common manipulation and well understood by those skilled in the art. In general, there are two methods that are used to complete this incorporation, and the necessary materials are available from various commercial sources (eg, Glen Research Inc., Sterling, Virginia, USA; Molecular Probes Inc., Eugene, Oregon , USA; TriLink BioTechnologies Inc., San Diego, California, USA; and others). The first method uses a fluorescent molecule that has been derivatized with a phosphoramidite radical to the phosphoramidite derivatives of the nucleosides described previously. In such a case, the fluorescent dye is attached to the polynucleotide attached to the support in the final chain assembly cycle. The fluorophore modified polynucleotide is then cleaved from the solid support and deprotected using the conventional procedures described above. This method has been called "direct marking". Alternatively, the second method uses a connective molecule derivatized with a phosphoramidite radical containing a protected reactive functional group (eg, amino, sulfhydryl, carbonyl, carboxyl, and others). This connecting molecule is attached to the polynucleotide attached to the support in the final cycle of the chain assembly. The modified polynucleotide with the linker is then cleaved from the solid support and deprotected using the conventional procedures described above. The functional group in the connector is deprotected either during the conventional deprotection procedure, or by the subsequent use of a specific treatment of the group. The modified polynucleotide with the crude linker is then reacted with an appropriate fluorophore derivative which will result in the formation of a covalent bond between a site in the fluorophore and the linker functional group. This method has been called "indirect marking."

Once synthesized, the polynucleotides of the present invention can be used immediately or stored for future use. Preferably, the polynucleotides of the invention are stored in duplex form in a suitable buffer. Many buffers are known in the art suitable for the storage of siRNAs. For example, the buffer may be formed by 100 mM KCl, 30 mM HEPES pH 7.5, and 1 mM MgCl2.

Preferably, the siRNAs of the present invention retain 30% to 100% of their activity when stored in such a buffer at 4 ° C for one year. More preferably, they retain 80% to 100% of their biological activity when stored in such a buffer at 4 ° C for one year. Alternatively, the compositions may be stored at -20 ° C in such a buffer for at least one year or more. Preferably, storage for a year or more at -20 ° C produces less than a 50% decrease in biological activity. More preferably, storage for a year or more at -20 ° C produces less than a 20% decrease in biological activity after a year or more. Most preferably, storage for a year or more at -20 ° C produces less than a 10% decrease in biological activity.

In order to guarantee the stability of the siRNA reserves before use, they can be kept in form at -20 ° C until they are ready for use. Before use, they must be resuspended; however, once resuspended, for example, in the above-mentioned buffer, they should be kept at -20 ° C until use. The aforementioned buffer, before use, can be stored at approximately 4 ° C

or at room temperature. The effective temperatures at which transfection is carried out are well known to those skilled in the art, and include, for example, room temperature.

In order to form the duplex siRNA from the complementary component strands, equal amounts of the effector strand and the antisense strand are mixed. Since oligonucleotides retain 2'ortoester protection at this point, they are treated with mild acid at 55 ° C, whose treatment eliminates these protecting groups. The unprotected strands hybridize to form the duplex allowing the deprotection solution to cool slowly to room temperature. Finally, the duplex is subjected to salt removal by precipitating it with ethanol, and the purified duplex is dissolved in RNase-free water and quantified by ultraviolet spectroscopy. The quality of the duplex formation process is evaluated by native gel electrophoresis.

The applications for this new and novel technology are wide. For example, it is possible for an individual to identify one or more targeted siRNAs, e.g. eg, against a therapeutically important target. Such a molecule could provide excellent silencing of the target of interest, but simultaneously silence additional genes (nonspecific targets) that have partial complementarity with the effector and / or antisense strand of the siRNA. The silencing of these secondary targets (nonspecific targets) can induce undesirable effects

(e.g., cell death, cell proliferation, differentiation) and for this reason, it is advantageous to eliminate the non-specific effects associated with the siRNA of interest.

Additionally, the siRNA of the present invention can be used in a variety of applications, including, but not limited to, basic research, drug discovery and development, diagnosis, and therapy. In research environments, the application may involve the introduction of modified molecules into cells using either the reverse transfection protocol or the direct transcription protocol. For example, the present invention can be used to validate whether a gene product is a target for the discovery or development of a drug. In this application, the mRNA that corresponds to a target nucleic acid sequence of interest is identified for degradation chosen as the target. The siRNAs of the invention that are specific for targeting the particular gene are introduced into a cell or organism, preferably in the form of a duplex. The cell or organism is maintained under conditions that allow degradation of the mRNA chosen as the target, resulting in a decrease in the activity or expression of the gene. The degree of any decreased expression or activity of the gene is then measured, together with the effect of said decreased expression or activity, and a determination is made as to whether the expression or activity decreases, in that case the nucleic acid sequence of interest It is an agent for drug discovery or development. In this way, phenotypically desirable effects can be associated with RNA interference of specific target nucleic acids of interest, and in appropriate cases toxicity and pharmacokinetic studies can be undertaken and therapeutic preparations developed.

The invention can be implemented for a wide range of applications associated with the transfection of modified molecules. In a non-limiting example, the modifications of the invention are used to eliminate the non-specific effects resulting from transfection of siRNA in a reverse transfection format. For example, the modified siRNAs are dried on a solid surface (e.g., the bottom of a well of a 96, 384, or 1536 well plate), solubilized by the addition of a carrier (e.g., a lipid transfection reagent), followed by the addition of cell types of choice for transfection.

In another application of the invention, siRNAs that are modified with the modifications of the invention, or unmodified, are transfected separately to the cells and executed element by element to identify or distinguish between the phenotypic results induced by the reduction of the gene expression that are generated by the specific silencing of the target and the silencing of nonspecific targets.

In another application of the use of modified siRNAs of the invention, cells are transfected with modified individual siRNA reserves or modified siRNAs that constitute the reserves. In this way, a user is able to identify the most functional siRNA or combination of siRNA against an individual target.

In yet another application, the siRNAs carrying the modifications of the invention are directed against a specific family of genes (e.g., kinases), genes associated with one or more specific routes (e.g., cell cycle regulation) , or complete genomes (eg, the human, rat, mouse C. elegans, or Drosophila genome). Reducing the expression of each gene in the collection with siRNA carrying the modifications of the invention would allow researchers to quickly assess the contribution of each member of a family of genes, or each member of a path, or each gene of a genome , to a specific biological function or event without the risk that the phenotype is the result of a nonspecific effect. As an example of this kind of application, individuals who are interested in identifying one or more host (human) genes that contribute to capacity, e.g. For example, if the HIV virus infects human cells, it can grow on siRNA plates directed against the entire human genome in an RTF format. After lipoplex formation, cells susceptible to HIV infection (eg, JC53 cells) are added to each well for transfection. After culturing the cells for a period of 24-48 hours, the cells in each well could be subjected to a lethal titer of the HIV virus. After an appropriate incubation period necessary for infection, plaques could be examined to identify which wells contain living cells. Wells that contain living cells (or a substantially larger number of living cells than controls) identify a host gene that is necessary for viral infection, replication, and / or release. In this way, host genes that play a role in pathogen infection can be identified with the risk that the observed phenotype is the result of nonspecific effects.

In yet another application, siRNA transfected cells bearing the modifications of the invention are used to evaluate the contribution of a particular gene (from the target) to the exclusion of a drug from the cells. In a non-limiting example, cells are subjected to reverse transfection on RTF plates containing one or more siRNAs directed against all known members of the human genome, siRNA directed against a specific family of genes (e.g., kinases), siRNA directed against genes of a specific route (eg, ADME-tox routes). Subsequently, the cells are treated with a specific compound (eg, a potential therapeutic compound) and the ability of the cells to be measured, e.g. eg, conserve, excrete, metabolize, or adsorb that compound and compare with untreated cells. Thus, a researcher can identify one or more host genes that play a role in the pharmacokinetics of the compound with a limited risk that the observed phenotype is the result of downregulation of a nonspecific gene.

In yet another application, siRNA transfected cells carrying the modifications of the invention are used to validate the target of one or more biologically relevant agents (eg, a drug). For example, if it is believed that a specific drug targets a specific protein and induces a specific phenotype, the action of the drug can be validated by locating its target protein with a specific siRNA of the gene that carries the modifications of the invention. If the siRNA induces the same phenotype as the drug, the target is validated. If the modified siRNA fails to induce the same phenotype, these experiments would question the validity of the protein as the drug's target.

In yet another application, two or more siRNAs bearing the modifications of the invention can be used and are directed to two or more different targets to identify and study synthetic lethal pairs.

In yet another application, the siRNAs bearing the modifications of the invention can be used to direct transcripts containing single nucleotide polymorphisms (SNPs) to facilitate and evaluate the contribution of a particular SNP to a phenotype, a biological function, a state of illness, or an event.

In yet another application, siRNA carrying the modifications of the invention can be used to target one or more target genes whose reduction in expression is known to induce a specific disease state. Thus, it is possible to facilitate the study of that specific disease without the risk of reducing the expression of additional genes.

In all the applications described above, the applications can be used in such a way that they reduce the expression of one or multiple genes in a single well.

The present invention can also be used in RNA interference applications that induce disease states or transient or permanent disorders in an organism, for example, by attenuating the activity of a target nucleic acid of interest that is believed to be a cause or factor in The disease or disorder of interest. Increasing the activity of a target nucleic acid of interest may make the disease or disorder worse, or tend to alleviate or cure the disease or disorder of interest, as the case may be. Similarly, decreasing the activity of the target nucleic acid of interest can make the disease or disorder worse.

or shop to relieve or heal, as the case may be. The target nucleic acids of interest may comprise genomic nucleic acids or extrachromosomal nucleic acids, such as viral nucleic acids.

Even more, the present invention can be used in RNA interference applications, such as diagnostic, prophylactic, and therapeutic applications of the compositions in the manufacture of a medicament in animals, preferably mammals, more preferably humans in the treatment of diseases. , or the excess or default expression of a target. Preferably, the disease or disorder is one that arises from the malfunction of one or more proteins, whose disease or disorder is related to the expression of the gene product of one or more proteins. For example, it is widely recognized that certain human breast cancers are related to the malfunction of a protein expressed from a gene commonly known as the "bcl-2" gene. A medicament can be made according to the compositions and teachings of the present invention, using one or more siRNAs directed against the bcl-2 gene, and optionally combining it with a pharmaceutically acceptable carrier, diluent and / or adjuvant, the medicament of which can be used. for the treatment of breast cancer. Applicants have established the usefulness of methods and compositions in cellular models. Methods of releasing polynucleotides, such as siRNA, to cells inside animals, including humans, are well known in the art. It is expected that any release vehicle currently known in the art, or that will be known, and has utility to introduce polynucleotides, such as siRNA, into animals, including humans, will be useful in the manufacture of a medicament according to the present invention, provided that the release vehicle is not incompatible with any of the modifications that may be present within the composition made in accordance with the present invention. A release vehicle that is not compatible with a composition made in accordance with the present invention is one that reduces the effectiveness of the composition by more than 95% measured against the efficacy in cell culture.

There are animal models for many, many disorders, including, for example, cancers, diseases of the vascular system, inborn errors of metabolism, and the like. It is within the practical knowledge of the art to administer nucleic acids to animals in dosing regimens until they reach an optimal dosage regimen for a particular disease or disorder in an animal such as a mammal, for example, a mouse, rat or primate. non-human. Once efficacy in the mammal has been established through routine experimentation by a person skilled in the art, dosage regimens can be achieved for the start of testing in humans based on the data obtained in such studies.

Dosages of medicaments manufactured in accordance with the present invention may vary from micrograms per kilogram to hundreds of milligrams per kilogram of a subject. As is known in the art, the dosage will vary according to the mass of the mammal receiving the dose, the nature of the mammal receiving the dose, the severity of the disease or disorder, and the stability of the medication in the subject's serum, among other factors well known to those skilled in the art.

For these applications, an organism that is suspected of having a disease or disorder that is susceptible to modulation by manipulating a specific target nucleic acid of interest is treated by administering siRNA. The results of siRNA treatment can be mitigating, palliative, prophylactic, and / or diagnoses of a specific disease or disorder. Preferably, the siRNA is administered in a pharmaceutically acceptable manner with a pharmaceutically acceptable carrier or diluent.

The therapeutic applications of the present invention can be made with a variety of therapeutic compositions and methods of administration. Pharmaceutically acceptable carriers and diluents are known to those skilled in the art. Methods of administration to cells and organisms are also known to those skilled in the art. It is known that dosing regimens, for example, depend on the severity and degree of sensitivity of the disease or disorder to be treated, with a course of treatment spanning days to months, or until the effect is achieved. desired about the disorder or disease status. Chronic administration of siRNA may be required for lasting desired effects with some diseases or disorders. Suitable dosage regimens can be determined, for example, by administering varying amounts of one or more siRNA in a pharmaceutically acceptable carrier or diluent, by means of a pharmaceutically acceptable release route, and the amount of drug accumulated in the organism of the recipient organism. It can be determined at different times after administration. Similarly, the desired effect (for example, the degree of suppression of the expression of a gene product or a gene activity) can be measured at different times after administration of the siRNA, and this data can be correlated with other data pharmacokinetics, such as accumulation in the organism or an organ. A person skilled in the art can determine the optimum dosages, dosage regimens, and the like. A person skilled in the art can use EC50 data from animal models in vivo and in vitro as guidelines for studies with humans.

Even more, the present invention can be used in RNA interference applications, such as diagnostic, prophylactic, and therapeutic applications. For these applications, an organism that is suspected of having a disease or disorder that is susceptible to modulation is treated by manipulating a particular target nucleic acid of interest through the administration of siRNA. The results of siRNA treatment can be mitigating, palliative, prophylactic, and / or diagnoses of a specific disease or disorder. Preferably, the siRNA is administered in a pharmaceutically acceptable manner with a pharmaceutically acceptable carrier and / or diluent.

Additionally, the siRNA can be administered in a topical cream or ointment, an oral preparation such as a capsule or tablet or a suspension or solution, and the like. The route of administration can be intravenous, intramuscular, dermal, subdermal, cutaneous, subcutaneous, intranasal, oral, rectal, by means of eye drops, by tissue implantation of a device that releases the siRNA at an advantageous location, for example near a organ or tissue or cell type that houses a target nucleic acid of interest.

Examples

Example 1

SiRNA synthesis

RNA oligonucleotides were synthesized using 2'-ACE chemistry (see Figure 3). The synthesis is preferably carried out in the form of an automated procedure in an appropriate machine. Several such synthesizing machines are known to those skilled in the art. Each nucleotide is added sequentially (3 'to 5' direction) to an oligonucleotide solid attached to the support. Although polystyrene supports are preferred, any suitable support can be used. The first nucleoside at the 3 'end of the strand is covalently anchored to a solid support. The nucleotide precursor, an activated ribonucleotide such as a phosphoramidite or H-phosphonate, and an activator such as tetrazole, for example, S-ethyl-tetrazole (although any other suitable activator can be used) are added (step i in Figure 3 ), coupling the second base to the 5 'end of the first nucleoside. The support is washed and any 5'-hydroxyl group that has not reacted with an acetylation reagent such as, but not limited to, acetic anhydride or phenoxyacetic anhydride to produce non-reactive 5'-acetyl radicals (step ii) is terminally protected. The P (III) bond is then oxidized to the most stable and ultimately desired P (V) link (step iii), using a suitable oxidizing agent such as, for example, t-butyl hydroperoxide or iodine and water. At the end of the nucleotide addition cycle, the 5'-silyl group is cleaved with fluoride ion (step iv), for example, using triethylammonium fluoride or t-butylammonium fluoride. The cycle is repeated for each subsequent nucleotide. It should be noted that, although Figure 3 illustrates a phosphoramidite having a methyl protecting group, any other suitable group can be used to protect or replace oxygen from the phosphoramidite radical. For example, alkyl groups, cyanoethyl groups, or thioderivatives can be used in this position. Additionally, the activated nucleoside entering step (i) may be a different type of activated nucleoside, for example, an H-phosphonate, methylphosphoramidite or a thiophosphoramidite. It should be noted that the initial nucleoside, or 3 ', attached to the support may have a different 5' protecting group such as a dimethoxytrityl group, rather than a silyl group. The cleavage of the dimethoxytrityl group requires acid hydrolysis, as used in conventional DNA synthesis chemistry. Therefore, an acid such as dichloroacetic acid (DCA) or trichloroacetic acid (TCA) is used for this step alone. In addition to the cleavage stage with DCA, the cycle is repeated as many times as necessary to synthesize the desired polynucleotide.

After synthesis, phosphate protecting groups, which are represented as methyl groups in Figure 3, but which do not have to be limited to methyl groups, are cleaved within 30 minutes using 2-carbamoyl-2-cyanoethylene-1 trihydrate, 1 M disodium dithiolate (dithiolate) in DMF (dimethylformamide). The deprotection solution is washed from the oligonucleotide bound to the solid support using water. The support is then treated with 40% methylamine for 20 minutes at 55 ° C. This releases the RNA oligonucleotides in solution, protects the exocyclic amines and eliminates the protection of acetyl in the 2'-ACE groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

The 2'-orthoester groups are the last protecting groups to be removed, if separation is desired. The structure of the 2'-ACE protected RNA immediately before the 2 'deprotection is shown in Figure 4.

For automated procedures, solid supports having the initial nucleoside are installed in the synthesizing apparatus. The apparatus will contain all accessory auxiliary reagents and monomers necessary for synthesis. The reagents are kept under an argon atmosphere, since some monomers, if not kept under an inert gas, can be hydrolyzed. The device is primed to fill all streets with the reagent. A synthesis cycle is designed that defines the release of the reagents in the proper order according to the synthesis cycle, releasing the reagents in a specified order. Once a cycle is defined, the amount of each reagent to be added is defined, the time between the stages is defined, and the washing steps are defined, the synthesis is ready to proceed once the support is added solid that has the initial nucleoside.

For the RNA analogs described herein, the modification is achieved through three different general methods. The first, which is implemented for carbohydrates and base modifications, as well as for the introduction of certain conjugated connectors and products, uses modified phosphoramidites in which the modification is pre-existing. An example of such a modification would be the 2'-modified carbohydrate species (2'-F, 2'-NH2, 2'-O-alkyl, etc.) wherein the 2 'orthoester is replaced by the desired modification; 3 'or 5' terminal modifications such as fluorescein derivatives, Dabsyl, cholesterol, cyanine derivatives or polyethylene glycol could also be introduced. Certain modifications would also be made to the internucleotide bond through the entry of the reactive nucleoside intermediate. Examples of the resulting internucleotide linkage modification include, but are not limited to methylphosphonates, phosphoramidates, phosphorothioates or phosphorodithioates.

Many modifiers can be used using the same or similar cycles. Examples of this class would include, for example, 2-aminopurine, 5-methylcytidine, 5-aminoalyluridine, diaminopurine, 2-O-alkyl, multi-atom spacers, individual spacer monomers, 2'-aminonucleosides, 2'-fluoronucleosides, 5 - iodouridine, 4-thouridine, acridines, 5-bromouridine, 5-fluorocytidine, 5-fluorouridine, 5-iodouridine, 5-iodocytidine, 5-biotin-thymidine, 5-fluorescein-thymidine, inosine, pseudouridine, abasic monomer, nebularan, desabulid, pyrenonucleoside, azanucleoside, etc. Often, the rest of the stages in the synthesis would remain the same, with the exception of modifications that introduce substituents that are labile to standard deprotection conditions. Here modified conditions would be used that do not affect the substituent. Second, certain modifications of the internucleotide bond require an alteration of the oxidation stage to allow its introduction. Examples of this class include phosphorothioates and phosphorodithioates in which oxidation with elemental sulfur or other suitable sulfur transfer agent is required. Third, certain conjugated products and modifications are introduced by the "post-synthesis" process, in which the desired molecule is added to the biopolymer after the solid phase synthesis is completed. An example of this would be the addition of polyethylene glycol to a pre-synthesized oligonucleotide containing a primary amine attached to a hydrocarbon linker. Anchoring in this case can be achieved by using a polyethylene glycol N-hydroxy-succinimidyl ester in a solution phase reaction.

While this outlines the most preferred method for the synthesis of synthetic RNA and its analogs, any method of nucleic acid synthesis that could assemble these molecules in their assembly could be employed. Examples of alternative methods include the 5'-DMT-2'-TBDMS and 5'-DMT-2'-TOM synthesis approaches. Some 2'-O-methyl, 2'-F and skeleton modifications may be introduced in the transcription reactions using modified and wild-type T7 and SP6 polymerases, for example.

Synthesis of modified RNA

The following guidelines are provided for the synthesis of modified RNAs, and can be easily adapted for use in any of the automated synthesizers known in the art.

3 'end modifications

There are several methods to incorporate 3 'modifications. The 3 'modification can be anchored or "loaded" on a solid support of choice using methods known in the art. Alternatively, the 3 'modification may be available in the form of a phosphoramidite. The phosphoramidite is coupled to a universal support, using conventional synthesis methods, where the universal support provides a hydroxyl in which a modification is created at the 3 'end by the introduction of the activated phosphoramidite of the desired terminal modification. According to another method, the 3 'modification could be introduced post-synthetically after the polynucleotide is removed from the solid support. The free polynucleotide initially has a 3 'terminal hydroxyl, amino, thiol, or halogen that reacts with an appropriately activated form of the modification of choice. Examples include, but are not limited to reactions with N-hydroxysuccinimidyl ester, thioether, disulfide, maleimido, or haloalkyl. This modification now becomes the 3 'end of the polynucleotide. Examples of modifications that can be conjugated post-synthetically can be, but are not limited to fluorosceins, acridines, TAMRA, dabsyl, cholesterol, polyethylene glycols, multi-atom spacers, cyanines, lipids, carbohydrates, fatty acids, steroids, peptides or polypeptides

5 'end modifications

There are several ways to introduce a 5 'modification into a polynucleotide. For example, a nucleoside having the 5 'modification can be purchased and subsequently activated to a phosphoramidite, or the phosphoramidite having the 5' modification may be commercially available. Next, the activated nucleoside having the 5 'modification in the cycle is used as any other activated nucleoside can be used. However, not all 5 'modifications are available as phosphoramidites. In such a case, the 5 'modification can be introduced in a manner analogous to that described for the previous 3' modifications.

Thioates

Polynucleotides having one or more thioate moieties, such as phosphorothioate linkages, were made according to the synthesis cycle described above and illustrated in Figure 3. However, instead of the oxidation stage with t-butyl hydroperoxide, elemental sulfur or other sulfurizing agent was used.

5'-uncle modifications

Monomers having 5 'thiols in the form of phosphoramidites can be purchased from commercial suppliers such as Glen Research. These 5 'thiol modified monomers generally carry trityl protecting groups. After synthesis, the trityl group can be removed by any method known in the art.

Other modifications

For certain modifications, the stages of the synthesis cycle may vary somewhat. For example, when the 3 'end has an inverse dT (where the first base is anchored to the solid support through the 5'-hydroxyl and the first coupling is a 3'-3' connection) the detrilation and coupling occur more slowly, so extra destritilant reagent, such as dichloroacetic acid (DCA) should be used, and the coupling time should be increased to 300 seconds. Some 5 'modifications may require prolonged coupling time. Examples include cholesterol, such as biotin Cy3 or Cy5 fluorophores, dabsyl, amino connectors, thio linkers, spacers, polyethylene glycol, phosphorylation reagent, BODIPY, or photosensitive connectors.

It should be noted that if a polynucleotide is going to have only a single modification, that modification can be carried out more efficiently manually by removing the support that the partially constructed polynucleotide has on it, manually coupling the monomer that has the modification, and then replacing the support in the automatic synthesizer and resuming automated synthesis.

Example 2

Deprotection and excision of oligonucleotides synthesized from the support

Excision can be performed manually or in an automatic procedure on a machine. The cleavage of the protective radical of the internucleotide connection, for example, a methyl group, can be achieved by the use of any suitable cleavage agent known in the art, for example, dithiolate or thiophene. 1 M dithiolate in DMF is added to the solid support at room temperature for 10 to 20 minutes. The support is thoroughly washed with, for example, DMF, then with water, then acetonitrile. Alternatively, a water wash followed by a thorough acetonitrile wash will be sufficient to remove any residual dithioate.

Excision of the support polynucleotide and removal of the protection of the exocyclic base can be performed with 40% aqueous N-methylamine (NMA), followed by heating at 55 degrees Celsius for twenty minutes. Once the polynucleotide is in solution, the NMA is carefully removed from the solid support. The solution containing the polynucleotide is then dried, to remove the NMA in vacuo. Further processing, including duplex formation, salt removal, gel purification, quality control, and the like can be carried out by any method known in the art.

For some modifications, the stage with NMA may vary. For example, for the modification of a 3 'amino, the treatment with NMA should be for forty minutes at 55 degrees Celsius. Puromycin, modifications of 5 'amino terminal connectors, and modifications of 2' amino nucleosides are heated for 1 hour after the addition of 40% NMA. Cy5 modified oligonucleotides are treated with ammonium hydroxide for 24 hours while being protected from light.

Preparation of cleavage reagents Quality water for HPLC and quality acetonitrile are used for synthesis. The dithiolate is previously prepared in the form of crystals. Add 4.5 grams of dithiolate crystals to 90 ml of DMF. NMA 40%, ready for use, can be purchased from a supplier such as Sigma Aldrich Corporation.

Annealing single stranded polynucleotides

Single stranded polynucleotides can be annealed by any method known in the art, using any suitable buffer. For example, equal amounts of each strand can be mixed in a suitable buffer, such as, for example, 50 mM HEPES pH 7.5, 100 mM potassium chloride, 1 mM magnesium chloride. The mixture is heated for one minute at 90 degrees Celsius, and allowed to cool to room temperature. In another example, each polynucleotide is prepared separately so that each is at a concentration of 50 micromolar. 30 µL of each polynucleotide solution is then added to a tube with 15 microliters of 5X annealing buffer, where the final concentration of the annealing buffer is 100 mM potassium chloride, 30 mM HEPES-KOH, pH 7.4 and 2 mM magnesium chloride. The final volume is 75 microliters. The solution is then incubated for one minute at 90 degrees Celsius, centrifuged in a centrifuge for 15 seconds, and allowed to incubate at 37 degrees Celsius for one hour, then allowed to reach room temperature. This solution can then be stored frozen at minus 20 degrees Celsius and freeze-thaw up to five times. The final concentration of the duplex is 20 micromolar. An example of a buffer suitable for polynucleotide storage is 20 mM KCl, 6 mM HEPES, pH 7.5, 0.2 mM MgCl2. All buffers used must be free of RNase.

Orthoester radical removal

If desired, the orthoester radical or radicals can be removed from the polynucleotide by any suitable method known in the art. One such method employs a pH 3.8 volatile acetic acid-tetramethylenediamine (TEMED) buffer system that can be removed by lyophilization after radical extraction

or orthoester radicals. Deprotection at a pH greater than 3.0 helps minimize the potential for acid catalyzed cleavage of the phosphodiester main strand. For example, deprotection can be achieved using 100 mM acetic acid adjusted to pH 3.8 with TEMED by suspending the orthoester protected polynucleotide and incubating it for 30 minutes at 60 degrees Celsius. The solution is then lyophilized or subjected to a SpeedVac until dry before use. If necessary, the removal of the salts can be carried out after deprotection by any method known in the art, for example, precipitation with ethanol or removal of the salts in a reverse phase cartridge.

Example 3

SiRNA synthesized for use in RNA interference

Nineteen unit siRNAs were synthesized that had a di-dT projection using ACE chemistry patented by Dharmacon, Inc.'s, and designed and used in accordance with the invention described herein. "SEAP" refers to human secreted alkaline phosphatase; "human cycle" refers to human cyclophilin B; an asterisk between nucleotide units refers to a modified internucleotide connection that is a phosphorothioate connection; the 2'-FC or 2'-FU structure refers to a nucleotide unit having a fluorine atom anchored to the 2 'carbon of a ribosyl radical; the 2'-N-C or 2'-N-U structure refers to a nucleotide unit having a -NH2 group attached to the 2 'carbon of a ribosyl radical; the structure of 2'-OMe-C or 2'-OMe-U refers to a nucleotide unit having a 2'-O-methyl modification in the 2 'carbon of a ribosyl radical of CS or Us, respectively; dG, dU, dA, dC, and dT refer to a nucleotide unit that is deoxy with respect to the 2 'position, and instead has a 2' carbon-anchored hydrogen of the ribosyl radical. Unless otherwise indicated, all nucleotide units in the following list are ribosyl with an OH in the 2 'carbon.

SiRNA duplex synthesis of general formula I

General Formula I:

S 5 '> HO-mX1MX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21-OH <3'

AS 3 '> HO-Y21Y20Y19Y18Y17Y16Y15Y14Y13Y12Y11Y10Y9Y8Y7Y6Y5Y4Y3Y2Y1-PO4 <5'

where Xq and Yq are ribonucleosides including rA, rC, rG, or rU; mXq are nucleosides with 2'-O-methyl including 2'-O-methyl-rA, 2'-O-methyl-rC, 2'-O-methyl-rG, and 2'-O-methyl-rU; S is the effector strand of the siRNA duplex; and AS is the antisense strand of the siRNA duplex.

Each strand (S and AS) of the duplex is chemically synthesized separately using the procedures described in US Patent No. 6,008,400; U.S. Patent No. 6,111,086; United States Patent No. 6,590,093; Scaringe (2000) Methods in Enzymology 317: 3-18; Scaringe (2001) Methods 23 (3): 206-217. Simply put, the procedures using a solid polystyrene support to which the nucleoside plus 3 '(X21 or Y21) has been covalently attached. The nucleosides are then sequentially added in a sequence specific manner (3 'to 5') to the species attached to the support using repetitive cycles. Therefore, the first cycle adds X20 to X21 or Y20 to Y21 and the second cycle adds X19 to X20X21 or Y19 to Y20Y21, and so on. Each cycle consists of four stages: deprotection of the 5'-hydroxyl group of the species bound to the support; coupling of a reactive derivative of the incoming nucleoside to the 5'-hydroxyl group of the species bound to the support; terminal protection of 5'-hydroxyl groups that have not reacted; and oxidation of the internucleotide connection. For Xq (q = 3 to 20) or Yq (q = 1 to 20) = a ribonucleoside, the reactive derivative is a 5'-silyl-2'ortoster-3'-phosphoramidite, in particular a 5'-O- Benzhydroxy-bis (trimethylsilyloxy) silyl-2'-O-bis (2-acetoxyethyl) orthoformyl-3'O- (N, N-diisopropyl) methylphosphoramidite (Figure 15).

Duplexes of General Formula I have 2'-O-methylnucleosides at positions 1 and 2 (mXq, q = 1 and 2) of the effector strand. These modified nucleosides are incorporated into S using 5'-silyl-2'-O-methyl-3'-phosphoramidites of appropriate sequence, in particular, 5'-O-benzhydroxy-bis (trimethylsilyloxy) -silyl-2'-O -methyl-3'-O- (N, N-diisopropyl) methylphosphoramidites (Figure 16), and the same reaction cycle used for ribonucleoside incorporation described above.

Duplexes of General Formula I have a phosphate radical at the 5 'end of the antisense strand. This phosphate group is chemically introduced using N, N-diisopropylamino-bis (2-cyanoethyl) phosphoramidite (Figure 17) and the same reaction cycle used for ribonucleoside incorporation described above.

After assembly of the strand, the fully protected oligonucleotide is treated with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate to remove methyl groups from internucleotide phosphate connections. The oligonucleotide is cleaved following the support and the base protecting groups and the 2-cyanoethyl groups in the 5'-phosphate are removed by treatment with aqueous N-methylamine solution, first at room temperature and then at 55 ° C, followed Vacuum drying At this point, the crude oligonucleotide is analyzed for quality by ion exchange HPLC and / or MALDI-TOF mass spectrometry, and gel purified, if necessary. The oligonucleotide is then dissolved in water or RNAse-free buffer and quantified by ultraviolet spectroscopy.

In order to form the duplex siRNA of the complementary strands of the components, equal amounts of the effector strand and the antisense strand are mixed. Since the oligonucleotides retain 2'ortoester protection at this point, they are treated with mild acid at 55 ° C, whose treatment eliminates these protecting groups. The unprotected strands are annealed to form the duplex by allowing the deprotection solution to cool slowly to room temperature. Finally, the duplex salts are removed by precipitating it with ethanol, and the purified duplex is dissolved in RNase-free water and quantified by ultraviolet spectroscopy. The quality of the duplex formation process was evaluated by native gel electrophoresis.

Synthesis of the siRNA duplex of General Formula II

  General Formula II

where

Xq and Yq are ribonucleosides including rA, rC, rG, or rU;

mXq and mYq are 2'-O-methylnucleosides including 2'-O-methyl-rA, 2'-O-methyl-rC, 2'-O-methyl-rG, and 2'-O-methyl-rU;

S is the effector strand of the siRNA duplex;

Y

AS is the antisense strand of the siRNA duplex.

Each strand (S and AS) of the duplex is chemically synthesized separately using the procedures described in US Patent No. 6,008,400; U.S. Patent No. 6,111,086; United States Patent No. 6,590,093; Scaringe (2000) Methods in Enzymology 317: 3-18; Scaringe (2001) Methods 23 (3): 206-217. Simply put, the procedures use a solid polystyrene support to which nucleosides plus 3 '(X21 or Y21) are covalently fixed. Nucleosides are then sequentially added in a sequence specific manner (3 'to 5') to the species attached to the support using repetitive cycles. Therefore, the first cycle adds X20 to X21 or Y20 to Y21; the second cycle adds X19 to X20X21 or Y19 to Y20Y21; and so on. Each cycle consists of four stages: deprotection of the 5'-hydroxyl group of the species bound to the support; coupling of a reactive derivative of the incoming nucleoside to the 5'-hydroxyl group of the species bound to the support; terminal protection of 5'-hydroxyl groups that have not reacted; and oxidation of the internucleotide connection. For Xq (q = 3 to 20) or Yq (q = 3 to 20) = a ribonucleoside, the reactive derivative is a 5'-silyl-2'-orthoester-3'phosphoramidite, in particular a 5'-O- Benzhydroxy-bis (trimethylsilyloxy) silyl-2'-O-bis (2-acetoxyethyl) orthoformyl-3'-O- (N, Ndiisopropyl) methylphosphoramidite (Figure 15).

Duplexes of General Formula II have 2'-O-methylnucleosides at positions 1 and 2 (mXq, q = 1 and 2) of the effector strand and at positions 1 and 2 (mXq, q = 1 and 2) of the antisense thread. These modified nucleosides are incorporated into S and AS using the 5'-silyl-2'-O-methyl-3'-phosphoramidites of appropriate sequence, in particular, 5'-Obenzhydroxy-bis (trimethylsilyloxy) -silyl-2'-O -methyl-3'-O- (N, N-diisopropyl) methylphosphoramidites (Figure 16), and the same reaction cycle used for ribonucleoside incorporation described above.

Duplexes of General Formula II have a phosphate radical at the 5 'end of the antisense strand. This phosphate group is chemically introduced using N, N-diisopropylamino-bis (2-cyanoethyl) phosphoramidite (Figure 17) and the same reaction cycle used for ribonucleoside incorporation described above.

After assembly of the strand, the fully protected oligonucleotide is treated with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate to remove methyl groups from internucleotide phosphate connections. The oligonucleotide is cleaved following the support and the base protecting groups and the 2-cyanoethyl groups in the 5'-phosphate are removed by treatment with aqueous N-methylamine solution, first at room temperature and then at 55 ° C, followed Vacuum drying At this point, the crude oligonucleotide is analyzed for quality by ion exchange HPLC and / or MALDI-TOF mass spectrometry, and gel purified, if necessary. The oligonucleotide is then dissolved in water or RNAse-free buffer and quantified by ultraviolet spectroscopy.

In order to form the duplex siRNA of the complementary strands of the components, equal amounts of the effector strand and the antisense strand are mixed. Since the oligonucleotides retain 2'ortoester protection at this point, they are treated with mild acid at 55 ° C, whose treatment eliminates these protecting groups. The unprotected strands are annealed to form the duplex by allowing the deprotection solution to cool slowly to room temperature. Finally, the duplex salts are removed by precipitating it with ethanol, and the purified duplex is dissolved in RNase-free water and quantified by ultraviolet spectroscopy. The quality of the duplex formation process was evaluated by native gel electrophoresis.

Synthesis of the siRNA duplex of General Formula III

  General Formula III

where

Xq and Yq are ribonucleosides including rA, rC, rG, or rU;

mXq and mYq are 2'-O-methylnucleosides including 2'-O-methyl-rA, 2'-O-methyl-rC, 2'-O-methyl-rG, and 2'-O-methyl-rU;

S is the effector strand of the siRNA duplex;

Y

AS is the antisense strand of the siRNA duplex.

Each strand (S and AS) of the duplex is chemically synthesized separately using the procedures described in US Patent No. 6,008,400; U.S. Patent No. 6,111,086; United States Patent No. 6,590,093; Scaringe (2000) Methods in Enzymology 317: 3-18; Scaringe (2001) Methods 23 (3): 206-217. Simply put, the procedures use a solid polystyrene support to which nucleosides plus 3 '(X21 or Y21) are covalently fixed. Nucleosides are then sequentially added in a sequence specific manner (3 'to 5') to the species attached to the support using repetitive cycles. Therefore, the first cycle adds X20 to X21 or Y20 to Y21; the second cycle adds X19 to X20X21 or Y19 to Y20Y21; and so on. Each cycle consists of four stages: deprotection of the 5'-hydroxyl group of the species bound to the support; coupling of a reactive derivative of the incoming nucleoside to the 5'-hydroxyl group of the species bound to the support; terminal protection of 5'-hydroxyl groups that have not reacted; and oxidation of the internucleotide connection. For Xq (q = 3 to 20) or Yq (q = 1 or 3 to 20) = a ribonucleoside, the reactive derivative is a 5'-silyl-2'-orthoester-3'phosphoramidite, in particular a 5'- O-benzhydroxy-bis (trimethylsilyloxy) silyl-2'-O-bis (2-acetoxyethyl) orthoformyl-3'-O- (N, Ndiisopropyl) methylphosphoramidite (Figure 15).

Duplexes of General Formula III have 2'-O-methylnucleosides at positions 1 and 2 (mXq, q = 1 and 2) of the effector strand and at positions 1 and 2 (mXq, q = 1 and 2) of the antisense thread. These modified nucleosides are incorporated into S and AS using the 5'-silyl-2'-O-methyl-3'-phosphoramidites of appropriate sequence, in particular, 5'-Obenzhydroxy-bis (trimethylsilyloxy) -silyl-2'-O -methyl-3'-O- (N, N-diisopropyl) methylphosphoramidites (Figure 16), and the same reaction cycle used for ribonucleoside incorporation described above.

Duplexes of General Formula III have a phosphate radical at the 5 'end of the antisense strand. This phosphate group is chemically introduced using N, N-diisopropylamino-bis (2-cyanoethyl) phosphoramidite (Figure 17) and the same reaction cycle used for ribonucleoside incorporation described above.

After assembly of the strand, the fully protected oligonucleotide is treated with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate to remove methyl groups from internucleotide phosphate connections. The oligonucleotide is cleaved following the support and the base protecting groups and the 2-cyanoethyl groups in the 5'-phosphate are removed by treatment with aqueous N-methylamine solution, first at room temperature and then at 55 ° C, followed Vacuum drying At this point, the crude oligonucleotide is analyzed for quality by ion exchange HPLC and / or MALDI-TOF mass spectrometry, and gel purified, if necessary. The oligonucleotide is then dissolved in water or RNAse-free buffer and quantified by ultraviolet spectroscopy.

In order to form the duplex siRNA of the complementary strands of the components, equal amounts of the effector strand and the antisense strand are mixed. Since the oligonucleotides retain 2'ortoester protection at this point, they are treated with mild acid at 55 ° C, whose treatment eliminates these protecting groups. The unprotected strands are annealed to form the duplex by allowing the deprotection solution to cool slowly to room temperature. Finally, the duplex salts are removed by precipitating it with ethanol, and the purified duplex is dissolved in RNase-free water and quantified by ultraviolet spectroscopy. The quality of the duplex formation process was evaluated by native gel electrophoresis.

Transfection

The siRNA duplexes were annealed using conventional buffer (50 millimolar HEPES pH 7.5, 100 milli molar KCl, 1 mM MgCl2). Transfections are performed according to the conventional protocol described below.

Conventional transfection protocol for 96-well and 6-well plates: siRNA

one.

 The protocols for 293 and Calu6, HeLa, MDA 75 are identical.

2.

 The cells are plated to be 95% confluent on the day of transfection.

3.

 SuperRNAsin (Ambion) is added to the transfection mixture for protection against RNAse.

Four.

 All solutions and manipulations must be carried out under RNase-free conditions.

Plate 1 0.5 -1 ml in 25 ml of medium in a small flask or 1 ml in 50 ml in a large flask.

96 well plate

one.

 Add 3 ml of 0.05% trypsin-EDTA in a medium flask (6 in a large flask) incubate 5 min at 37 degrees C.

2.

 Add 7 ml (14 ml in large) of regular medium and pipette 10 times up and down to resuspend the cells.

3.

 Take 25 microliters of the cell suspension from stage 2 and 75 microliters of trypan blue dye (1: 4) and place 10 microliters in a cell counter.

Four.

 Count the number of cells in a conventional hemocytometer.

5.

 The average number of cells x 4 x 10000 is the number of cells per ml.

6.

 Dilute with regular medium to have 350,000 / ml.

7.

 Cultivate 100 microliters (35,000 cells for HEK293) in a 96-well plate.

Transfection for 2 x 96 well plates (60 well format)

one.

 OPTI-MEM 2 ml + 80 microliters of Lipofectamine 2000 (1:25) + 15 microliters of SuperRNAsin (AMBION).

2.

 Transfer aliquots of siRNA (0.8 microliters of 100 micromolar for screening (the total dilution factor is 1: 750, 0.8 microliters of 100 micromolar solution will produce 100 final nanomolar) the deep source in a desired order (usually 3 x columns 6 for the format of 60 wells or four columns by 8 for 96 wells).

3.

 Transfer 100 microliters of OPTI-MEM.

Four.

 Transfer 100 microliters of OPTI-MEM with Lipofectamine 2000 and SuperRNAsin to each well.

5.

 Leave for 20-30 min at RT.

6.

 Add 0.55 ml of regular medium to each well. Cover the plate with plastic film and mix.

7.

 Arrange in cells 100 x 3 x 2 directly the cells (enough for two plates).

Transfection for 2 x 6 well plates

8.

 8 ml OPTI-MEM + 160 microliters Lipofectamine 2000 (1:25). 30 microliters of SuperRNAsin (AMBION).

9.

 Transfer aliquots of siRNA (the total dilution factor is 1: 750, 5 microliters of 100 micromolar solution will produce 100 nanomolar final) to polystyrene tubes.

10.

 Transfer 1,300 microliters of OPTI-MEM with Lipofectamine 2000 and SuperRNAsin (AMBION).

eleven.

 Leave for 20-30 min at RT.

12.

Add 0.55 ml of regular medium to each well. Cover the plate with plastic film and mix.

13.

 Transfer 2 ml to each well (enough for two wells).

The mRNA or protein levels are measured 24, 48, 72, and 96 hours after transfection with conventional kits

or sets of Custom B-DNA and Quantigene kits (Bayer).

Example 5

Activity measurement / detection

The level of RNA interference induced by siRNA, or gene silencing, was estimated by analyzing the reduction of the target mRNA levels or the reduction of the corresponding protein levels. Analysis of mRNA levels was carried out using B-DNA ™ technology (Quantagene Corp.). Protein levels for fLUC and rLUC were analyzed using STEADY GLO ™ kits (Promega Corp.). Human alkaline phosphatase levels were analyzed by Great EscAPe SEAP Fluorescence Detection Kits (No. K2043-1), BD Biosciences, Clontech.

For microarray analysis: HeLa cells were transfected in 6-well plates using Oligofectamine (Invitrogen) and the indicated doses of siRNA duplex. When not specified, the concentration of siRNA was 100 nM. RNA was isolated 24 hours after transfection. The RNA of the transfected cells with siRNA was hybridized against the RNA of simulated transfected cells (treated with transfection reagent in the absence of RNA duplex). Total RNA was purified by the Qiagen RNeasy kit, and processed for hybridization of microarrays containing oligonucleotides corresponding to approximately 21,000 human genes. Proportional hybridizations were performed with an inverse fluorescent label to eliminate dye bias. The microarrays were purchased from Agilent Technologies or synthesized as described by Hughes, TR, et al. (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat. Biotech. 19: 342-347. Each row represents the expression pattern resulting from the transfection of an individual siRNA. The microarray data are presented in Figures 7-11. The data shown are signature genes that show a difference in the level of expression (p value <0.01 and log10 intensity> -1.5) with respect to simulated transfected cells. No cuts were placed on the change in multiplicity of expression. The green color indicates a decrease in expression; The red color indicates an increase in expression. Data were analyzed using Rosetta Resolver ™ software. The histogram at the top of the cluster diagrams reflects the similarity of gene expression changes between the different genes analyzed in the experiment.

Example 6

Identification of chemical modifications that modify the silencing activity

Using the 2'-O-ACE chemistry as a platform for RNA synthesis, a modification walk consisting of one, two, or three consecutive modified nucleotides in the effector (S) and antisense (AS) strands was performed in SEAP-2217, an siRNA directed against human secreted alkaline phosphatase (SEAP, SEAP-2217 effector thread 5'-GUGAUGUAUGUCAGAGAGUdTdT-3 '(SEQ ID NO. 22). Subsequently, the silencing efficacy of these modified siRNAs was evaluated by cotransfection of each duplex with a SEAP expression vector (Clontech) in HEK293 cells (100 nM siRNA, 50 ng / well of SEAP expression vector, Lipofectamine 2000) and analyzing the decrease in target protein activity twenty four hours after Transfection Figure 5 shows the ratio between modification and function of SEAP-2217 2'-Omethylated siRNA Unmodified duplexes directed to SEAP induce> 90% silencing of the SEAP gene. A single base of both S and SA strands induced little or no effect on the activity of the siRNA, suggesting that no individual 2'-hydroxyl group in either strand plays an indispensable role in the specific RNAi of the target. In contrast, a walk of modifications, element by element, doubles identified several key positions in which the introduction of modified bases significantly interfered with the silencing activity. The deepest interference in the function was observed when two consecutive bases (positions 1 and 2) or three consecutive bases (positions 1, 2 and 3) of the 5 'end of the AS strand were modified, thus giving indications of an effect cooperative between adjacent modified groups. Since similar modifications of the S strand failed to alter the functionality of the duplex, the paired 2'O-methyl modified bases allow a distinction of the S and AS strands and the identification of the key positions to reduce the expression of the target. On the other hand, these experiments identify positions within the duplex that play a key role in silencing the target (and possibly non-specific targets).

Example 7

Additional analysis of the effects of combinations of different chemical modifications on siRNA-induced gene silencing

To test the effects of 2'-O-methyl modifications on duplex functionality in various combinations, a series of siRNAs directed against the luciferase gene were synthesized (luc 8, 18, 56, 58, 63, and 81) using 2'-O-ACE chemistry and were modified to contain O-methyl groups at the 2 'position of the ribose ring.

Luc 8 5'-GAAAAAUCAGAGAGAUCCU-3 '(SEQ ID NO. 23)

Luc 18 5'-UACCGGAAAACUCGACGCA-3 '(SEQ ID NO. 24)

Luc 56 5'-ACGUCGCCAGUCAAGUAAC-3 '(SEQ ID NO. 25)

Luc 58 5'-GAUUACGUCGCCAGUCAAG-3 '(SEQ ID NO. 26)

Luc 63 5'-AGAGAUCGUGGAUUACGUC-3 '(SEQ ID NO. 27)

Luc 81 5'-UGUUGUUUUGGAGCACGGA-3 '(SEQ ID NO. 28)

(The sequences mentioned above are the effector strand.)

Specifically, siRNAs containing 2'-O-methyl modifications were co-transfected in the two nucleotides plus 5 'of (1) the effector strand, (2) the antisense strand, or (3) both strands, together with an expression plasmid of Luc (pCMVLuc, 50 ng / well) in HEK293 cells. Subsequently, an element-by-element comparison of the silencing capacity of each duplex was performed to determine the effects of this modification on the degradation of the target transcript.

The results of these studies showed that the addition of 2'-O-methyl groups only to the AS strand dramatically decreased the ability of duplexes to silence the target mRNA (see Figure 6). In contrast, the duplexes that carry this modification in the effector strand also performed (Luc 58, 63, 81) or better (Luc 56, 8, 18) than the equivalent, the unmodified siRNA, suggesting that the modification of the effector strand inclined the strand selection by RISC and (in some cases) increased the effective concentration of the antisense strand. Improved silencing could be the result of a decrease in the binding affinity of RISC to the 5 'effector end of the molecule (and therefore an increase in the availability of free RISC for association at the opposite end), the decrease in ability of native kinases to phosphorylate the effector strand (thus decreasing competition between the effector and antisense strand for access to RISC), or a decrease in RISC's ability to untangle the duplex from the effector end 5 '. The siRNA containing modifications with 2'-O-methyl in both strands showed a decrease in silencing capacities that were among the values observed for molecules containing modifications in any individual strand. An interpretation of these results is that modifications with 2'-O-methyl reduce the binding affinity that RISC has for the modified strand. In cases where the two strands are modified, neither strand receives an advantage over its complement, and a new equilibrium is established that represents an average of the functionality of both modified molecules.

To test whether the decrease in the level of silencing observed in cells containing S / AS 2'-O-methylated siRNA was the result of a weakened ability of cell kinases to phosphorylate duplexes, the siRNAs that carry modifications with 2'-O-methyl was modified to carry a phosphate group at the 5 'end of the AS strand. Specifically, Luc siRNAs bearing 2'-O-methyl groups in any of: (1) positions 1 and 2 of the 5 'end of the antisense strand; or (2) positions 1 and 2 of the 5 'end of both effector and antisense strands were phosphorylated at 5' in the AS strand during synthesis. These duplexes were then introduced into HEK293 cells using the procedures described above and tested to determine the ability to silence the desired target. The results showed that in 83% of the cases tested (10/12), the 5 'phosphorylation of the antisense strand improved the effectiveness of duplex silencing on the equivalent non-phosphorylated molecule (Figure 6). In the remaining two cases, the silencing remained unchanged or only marginally improved. These results demonstrate that the combination of the 5 'phosphorylation of the antisense strand and the 2'O-methylation of positions 1 and 2 of the effector and antisense strands are compatible with the maintenance of duplex functionality. On the other hand, since the dual 2'-O-methyl modifications of positions 1 and 2 of a strand (in the absence of 5 'phosphorylation of the terminal position) severely compromise the silencing ability of the nonphosphorylated strand, this modification pattern (2'-O-methyl modification of positions 1 and 2 of the effector strand, 2'-Omethyl modification of positions 1 and 2 of the antisense strand, plus 5 'phosphorylation of the antisense strand ) identifies a strategy for the elimination of nonspecific targets of the effector strand without compromising the reduction of the expression of the nonspecific target. On the other hand, the potential effect of 2'-O-methylation on other stages led the authors to consider that the possibility that such modifications could also alter the ability of RISC to distinguish between the intended targets that have 100% homology with the antisense strand and nonspecific targets that have lower amounts of homology.

Example 8

Identification of chemical modification patterns that eliminate, minimize or alter the nonspecific effects generated by siRNA

To determine whether the siRNA containing the modification pattern (2'-O-methyl modification of positions 1 and 2 of the effector strand, 2'-O-methyl modification of positions 1 and 2 of the antisense strand, plus 5 'phosphorylation of the antisense strand) had the same effects on the nonspecific target or altered effects, siRNAs directed to IGFR1 (IGFR1-73) were transfected into cells in unmodified and modified states. As shown in Figure 7, although the unmodified version of the siRNA induced significant non-specific gene modulation, the modified form downregulated a much more limited subset. These findings were constantly observed through a wide range of siRNAs tested (See Figure 8 for the heat map of MAPK14-153 and MPHOSH1-202, and Figure 9 for the sum of the results in 8 different siRNAs directed to 4 genes). In all these cases, silencing by the fully modified molecule was more or less equivalent to the unmodified molecule.

To determine whether the number or position of the 2'-O-methyl modifications were important for the increase in specificity observed, the authors of the present invention made a chemical modification walk through MAPK14-153. All duplexes in these studies with the exception of duplex D (unmodified duplex) and duplex F (modified with 2'-O-methyl in positions 1 and 2 of the antisense strand, without modification in the antisense strand) contain modifications with 2'-O-methyl paired in positions 1 and 2 of the effector strand. In addition, all duplexes in this study (D → R) contain a phosphate group at the 5 'end of the AS strand. In addition, the complementary thread in the remaining configurations (E, G → Q) contains the following modifications:

E: 2'O-methyl modification of positions 1 and 2 of the AS strand

G: 2'O-methyl modification of positions 2 and 3 of the AS strand

H: 2'O-methyl modification of positions 3 and 4 of the AS strand

I: 2'O-methyl modification of positions 4 and 5 of the AS strand

J: 2'O-methyl modification of positions 5 and 6 of the AS strand

K: 2'O-methyl modification of positions 6 and 7 of the AS strand

L: 2'O-methyl modification of positions 7 and 8 of the AS strand

M: 2'O-methyl modification of positions 8 and 9 of the AS strand

N: 2'O-methyl modification of positions 9 and 10 of the AS strand

O: 2'O-methyl modification of positions 10 and 11 of the AS strand

P: 2'O-methyl modification of position1 of the AS strand

Q: 2'O-methyl modification of position2 of the AS strand

As shown in Figure 10a, only three modification patterns, E, G, and Q showed significant reductions in nonspecific effects. Since the common element between these three molecules is the modification at position 2 of the antisense strand, this position is identified as a key element for the elimination of nonspecific targets.

To confirm this discovery, three additional siRNAs designed for MAPK14, KNTC2, and STK6 were designed to contain: (1) changes in positions 1 and 2 of the effector strand; (2) the modifications in positions 1 and 2 of the effector strand plus the modifications in position 1 of the antisense strand; (3) the modifications in positions 1 and 2 of the effector strand plus the modifications in position 2 of the antisense strand; (4) the modifications in positions 1 and 2 of the effector strand plus the modifications in positions 1 and 2 of the antisense strand. The nonspecific effects generated by these molecules were compared with unmodified siRNA. In all cases studied, the antisense strand also contains a 5'-carbon phosphate group of the 5'-terminal nucleotide. The siRNAs directed to MAPK14, KNTC2, and STK6 (MAPK14, 5 '193 CCUACAGAGAACUGCGGUU-3' (SEQ. ID NO. 29), effector sequence; KNTC2, 5 'GGCUUCCUUACAAGGAGAU3' (SEQ. ID NO. 30), effector sequence; and STK6, 5 'CGGGUCUUGUGUCCUUCAA-3' (SEQ. ID NO. 31), effector sequence) show all significant levels of nonspecific effects when not modified (Figure 10b). In contrast, the addition of the following modification pattern: 2'-O-methyl modification of effector nucleotides 1 and 2, plus 2'-O-methyl modification of antisense nucleotides 1 and 2 (or 2), plus phosphorylation of the 5 'carbon of the first antisense nucleotide, was sufficient to eliminate most of the nonspecific effects. These studies demonstrate the fundamental importance of position 2 in limiting the nonspecific effects and the ability of the chemical modification pattern described in embodiment 1 to reduce and / or eliminate these effects.

Example 9

Evaluation of base mismatches to eliminate nonspecific effects: A comparison with chemically modified siRNA

To further explore the importance of position 2 in nonspecific effects, erroneous base pairings were incorporated into the siRNA directed to the MAPK14 gene. Duplexes that carry a single mismatch of base pairs (between the antisense strand and the target site of the target) were then compared with the siRNA that carried paired chemical modifications (2'-O-methyl modification) at positions a along the molecule (that is, positions 1 and 2, 2 and 3, 3 and 4, etc ... of the antisense strand). The results of these experiments are provided in Figure 11 and demonstrate several important points. First, as previously noted, the chemical modification of positions 1 and 2 has the greatest effect on the elimination of nonspecific signature, while modifications with 2'-O-methyl paired at other positions provided smaller amounts of unspecific silencing. Surprisingly, the introduction of erroneous pairings of base pairs in various positions along the duplex provided variable results, depending on the position of the pairing. A pairing at position 1 failed to eliminate the unspecific signature of the unmodified molecule and led to further / more downward regulation of some of the genes. The introduction of mismatches of base pairs at positions 2-7 eliminated a substantial portion of the signature generated by the unmodified duplexes, but frequently led to altered expression patterns of genes other than the target gene. For MAPK14-153, this was particularly evident when the wrong pairings were introduced at position 4 of the antisense strand. Together, these studies demonstrate that while both the mismatches of base pairs and the pattern of chemical modifications of claim 1 can alter the nonspecific effects of siRNA, the patterns of chemical modifications are superior due to the fact that a secondary signature It does not replace the pattern observed in unmodified molecules.

Example 10

Demonstration that the nonspecific effects of siRNA generate observable phenotypes: toxic siRNA

The importance of the present invention became apparent when it was recognized that nonspecific effects can induce phenotypes that were not associated with the reduction of target expression. This phenomenon was evident in a study of nonspecificity and cellular toxicity induced by siRNA. A randomly selected population of siRNA derived from a walk with siRNA directed to DBI (NM_020548, position 202-291) was evaluated to determine the ability to induce toxicity. The directed siRNA collection consisted of 90 individual duplexes (19 nt) and covered the respective region in single-base stages. The duplexes were transfected into HeLa cells (10.00 cells per well, 10nM siRNA) using Lipofectamine 2000 (Invitrogen) and a 75% cell viability threshold was used as an arbitrary cut to distinguish non-toxic toxic sequences. Cell survival after treatment was determined by cytotoxicity analysis with Alamar Blue (BioSource Int.) According to the manufacturers instructions.

It was observed that siRNAs transfected under these conditions induced varying levels of cell cytotoxicity. Overall, it was found that 14 of 90 siRNA duplexes (15.5%) decreased cell viability below 75% (Figure 12a). As an example that both toxic and non-toxic siRNAs could be found to induce a strong silencing of DBI, the relative cytotoxicity of each siRNA was not related to the reduction of the specific expression of the target.

Independent confirmation of the toxicity induced by siRNA was obtained from the analysis of a separate collection of 48 functional siRNAs (silencing> 70%) targeting 12 different genes ARAF1, NMA_001654, MAP2K1, NM_002755, MAP2K2, NMA_030662, PI3K-CA, NMA_006218, Pi3K-CB, NMA_006219 Bcl2, NM_000633, Bcl3, NM_005178, MAPK1, NM_002745, MAPK3, NM_002746, AR, NM_000044, SRD5a1, NM_001047, SRD5a2, NMA_000348, four ARNip. Only twelve of the forty-eight sequences (25%) reduced cell viability below 75%. An illustrative duplex group of this collection is shown in Figure 12c. Although the eight duplexes directed to MAPK1 and MAPK2 show a gene silencing greater than 80%, only a single siRNA of each quartet reduces cell viability below 75% (MAPK1-d4 and MAPK2-d3). Thus, since the remaining siRNAs of each group were equally functional in the ability to silence the target, but not toxic, the toxicity induced by MAP2K1-d4 and MAP2K2-d3 is not related to the reduction of target expression. . In addition, the level of relative toxicity was found to be dependent on the concentration of siRNA during transfection (Figure 12d). Since both toxicity and nonspecific effects induced by siRNA show a dependence on the concentration of siRNA, it was predicted that toxicity induced by siRNA was a nonspecific effect.

The linear presentation of the distribution of the toxic siRNA together with the walk through DBI showed that the dispersion of these sequences was frequently non-random (that is, grouped) and suggested the presence of one or more motifs that were responsible for the observed toxicity (Figure 12a, areas delimited in boxes). Subsequent analysis of the toxic sequences of the randomized functional siRNA set revealed that the twelve sequences contained an AAA / UUU or GCCA / UGGC motif. To test whether there was a correlation between the presence of these motifs and toxicity, three groups of randomly selected siRNAs were selected that contained AAA / UUU motifs, GCCA / UGGC motifs, or no motif, and were tested. to determine the ability to induce cell death. As shown in Figures 13a and 13b, the siRNA containing the AAA / CTUU and GCCA / UGGC motifs showed a higher probability of inducing toxicity (56% and 53%, respectively) than the non-motive siRNA (Figure 13c, 6%). Since the p-value of the T test for these two samples was 1.3 x 10-7, these findings strongly support the notion that there is a strong correlation between siRNA-induced cellular toxicity and duplex release containing AAA motifs. / UUU or GCCA / UGGC. The target sequences for the siRNAs used to generate the data in Figure 12 and 13 are provided in the Table.

I. Modifications of the corresponding siRNAs, as used, are indicated in the examples and the descriptions of the figures.

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

12A

NM_020548 DBI

one ACGGGCAAGGCCAAGUGGG 32

2 CGGGCAAGGCCAAGUGGGA 33

3 GGGCAAGGCCAAGUGGGAU 3. 4

4 GGCAAGGCCAAGUGGGAUG 35

5 GCAAGGCCAAGUGGGAUGC 36

6 CAAGGCCAAGUGGGAUGCC 37

7 AAGGCCAAGUGGGAUGCCU 38

8 AGGCCAAGUGGGAUGCCUG 39

9 GGCCAAGUGGGAUGCCUGG 40

10 GCCAAGUGGGAUGCCUGGA 41

eleven CCAAGUGGGAUGCCUGGAA 42

12 CAAGUGGGAUGCCUGGAAU 43

13 AAGUGGGAUGCCUGGAAUG 44

14 AGUGGGAUGCCUGGAAUGA Four. Five

fifteen GUGGGAUGCCUGGAAUGAG 46

16 UGGGAUGCCUGGAAUGAGC 47

17 GGGAUGCCUGGAAUGAGCU 48

18 GGAUGCCUGGAAUGAGCUG 49

19 GAUGCCUGGAAUGAGCUGA fifty

twenty AUGCCUGGAAUGAGCUGAA 51

twenty-one UGCCUGGAAUGAGCUGAAA 52

22 GCCUGGAAUGAGCUGAAAG 53

2. 3 CCUGGAAUGAGCUGAAAGG 54

24 CUGGAAUGAGCUGAAAGGG 55

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

25 UGGAAUGAGCUGAAAGGGA 56

26 GGAAUGAGCUGAAAGGGAC 57

27 GAAUGAGCUGAAAGGGACU 58

28 AAUGAGCUGAAAGGGACUU 59

29 AUGAGCUGAAAGGGACUUC 60

30 UGAGCUGAAAGGGACUUCC 61

31 GAGCUGAAAGGGACUUCCA 62

32 AGCUGAAAGGGACUUCCAA 63

33 GCUGAAAGGGACUUCCAAG 64

3. 4 CUGAAAGGGACUUCCAAGG 65

35 UGAAAGGGACUUCCAAGGA 66

36 GAAAGGGACUUCCAAGGAA 67

37 AAAGGGACUUCCAAGGAAG 68

38 AAGGGACUUCCAAGGAAGA 69

39 AGGGACUUCCAAGGAAGAU 70

40 GGGACUUCCAAGGAAGAUG 71

41 GGACUUCCAAGGAAGAUGC 72

42 GACUUCCAAGGAAGAUGCC 73

43 ACUUCCAAGGAAGAUGCCA 74

44 CUUCCAAGGAAGAUGCCAU 75

Four. Five UUCCAAGGAAGAUGCCAUG 76

46 UCCAAGGAAGAUGCCAUGA 77

47 CCAAGGAAGAUGCCAUGAA 78

48 CAAGGAAGAUGCCAUGAAA 79

49 AAGGAAGAUGCCAUGAAAG 80

fifty AGGAAGAUGCCAUGAAAGC 81

51 GGAAGAUGCCAUGAAAGCU 82

52 GAAGAUGCCAUGAAAGCUU 83

53 AAGAUGCCAUGAAAGCUUA 84

54 AGAUGCCAUGAAAGCUUAC 85

55 GAUGCCAUGAAAGCUUACA 86

56 AUGCCAUGAAAGCUUACAU 87

57 UGCCAUGAAAGCUUACAUC 88

58 GCCAUGAAAGCUUACAUCA 89

59 CCAUGAAAGCUUACAUCAA 90

60 CAUGAAAGCUUACAUCAAC 91

61 AUGAAAGCUUACAUCAACA 92

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

62 UGAAAGCUUACAUCAACAA 93

63 GAAAGCUUACAUCAACAAA 94

64 AAAGCUUACAUCAACAAAG 95

65 AAGCUUACAUCAACAAAGU 96

66 AGCUUACAUCAACAAAGUA 97

67 GCUUACAUCAACAAAGUAG 98

68 CUUACAUCAACAAAGUAGA 99

69 UUACAUCAACAAAGUAGAA 100

70 UACAUCAACAAAGUAGAAG 101

71 ACAUCAACAAAGUAGAAGA 102

72 CAUCAACAAAGUAGAAGAG 103

73 AUCAACAAAGUAGAAGAGC 104

74 UCAACAAAGUAGAAGAGCU 105

75 CAACAAAGUAGAAGAGCUA 106

76 AACAAAGUAGAAGAGCUAA 107

77 ACAAAGUAGAAGAGCUAAA 108

78 CAAAGUAGAAGAGCUAAAG 109

79 AAAGUAGAAGAGCUAAAGA 110

80 AAGUAGAAGAGCUAAAGAA 111

81 AGUAGAAGAGCUAAAGAAA 112

82 GUAGAAGAGCUAAAGAAAA 113

83 UAGAAGAGCUAAAGAAAAA 114

84 AGAAGAGCUAAAGAAAAAA 115

85 GAAGAGCUAAAGAAAAAAU 116

86 AAGAGCUAAAGAAAAAAUA 117

87 AGAGCUAAAGAAAAAAUAC 118

88 GAGCUAAAGAAAAAAUACG 119

89 AGCUAAAGAAAAAAUACGG 120

90 GCUAAAGAAAAAAUACGGG 121

12B

NM_000633 Bcl2 Bcl2 2 GAAGUACAUCCAUUAUAAG 122

NM_002745 MAPK1 MAPK1 2 AAACAGAUCUUUACAAGCU 123

NM_006219 PI3K Cb PI3K Cb 4 UUUCAAGUGUCUCCUAAUA 124

NM_001654 ARaf1 Raf1 2 GCAAAGAACAUCAUCCAUA 125

NM_002755 MAP2K1 MAP2K1 2 GCAGAGAGAGCAGAUUUGA 126

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

NMA_000044 AR AR 3 UCAAGGAACUCGAUCGUAU 127

NMA_001654 ARaf1 Raf1 3 GACAUGAAAUCCAACAAUA 128

NM_006219 PI3K Cb PI3K Cb 2 UCAAGUGUCUCCUAAUAUG 129

NM_030662 MAP2K2 MAP2K2 1 CAAAGACGAUGACUUCGAA 130

NM_030662 MAP2K2 MAP2K2 4 GGAAGCUGAUCCACCUUGA 131

NM_002745 MAPK1 MAPK1 3 CAAGAGGAUUGAAGUAGAA 132

NM_002745 MAPK1 MAPK1 1 CCAAAGCUCUGGACUUAUU 133

NM_000633 Bcl2 Bcl2 3 GUACGACAACCGGGAGAUA 134

NM_000633 Bcl2 Bcl2 4 AGAUAGUGAUGAAGUACAU 135

NM_000633 Bcl2 Bcl2 1 GGGAGAUAGUGAUGAAGUA 136

NM_000044 AR AR 4 GAAAUGAUUGCACUAUUGA 137

NM_001654 ARaf1 Raf1 1 GCACGGAGAUGUUGCAGUA 138

NM_000348 SRD5A2 SRD5A2 1 GCUACUAUCUGAUUUACUG 139

NM_000044 AR AR 2 CAAGGGAGGUUACACCAAA 140

NM_002755 MAP2K1 MAP2K1 3 GAGGUUCUCUGGAUCAAGU 141

NM_002746 MAP3 MAPK3 4 GCUACACGCAGUUGCAGUA 142

NM_002746 MAPK3 MAPK3 2 AGACUGACCUGUACAAGUU 143

NM_030662 MAP2K2 MAP2K2 2 GAUCAGCAUUUGCAUGGAA 144

NM_000044 AR AR-1 GGAACUCGAUCGUAUCAUU 145

NM_006219 PI3K Cb PI3K Cb-1 CGACAAGACUGCCGAGAGA 146

NM_005178 Bcl3 Bcl3 2 GAGCCUUACUGCCUUUGUA 147

NM_006218 PI3K Ca PI3K Ca 4 CUGAAGAAAGCAUUGACUA 148

NM_005178 Bcl3 Bcl3 3 GGCCGGAGGCGCUUUACUA 149

NM_002745 MAPK1 MAPK1 4 GUACAGGGCUCCAGAAAUU 150

NM_005178 Bcl3 Bcl3 4 UCGACGCAGUGGACAUUAA 151

NM_006218 PI3K Ca PI3K Ca 2 AACUAGAAGUAUGUUGCUA 152

NM_002746 MAPK3 MAPK3 1 GACCGGAUGUUAACCUUUA 153

NM_000348 SRD5A2 SRD5A2 4 UUGGGUGUCUUCUUAUUUA 154

NM_006218 PI3K Ca PI3K Ca 3 AAUGGCUUUGAAUCUUUGG 155

NM_002755 MAP2K1 MAP2K1 1 GCACAUGGAUGGAGGUUCU 156

NM_006219 PI3K Cb PI3K Cb 3 GGAUUCAGUUGGAGUGAUU 157

NM_001654 ARaf1 Raf1 4 CAAAGAACAUCAUCCAUAG 158

NM_001047 SRD5A1 SRD5A1 3 GAAAGCCUAUGCCACUGUU 159

NM_000348 SRD5A2 SRD5A2 2 GCUAUGCCCUGGCCACUUG 160

NM_002755 MAP2K1 MAP2K1 4 GAGCAGAUUUGAAGCAACU 161

NM_001047 SRD5A1 SRD5A1 2 UAACUGCAGCCAACUAUUU 162

NM_006218 PI3K Ca PI3K Ca-1 AUGUUUACUACCAAAUGGA 163

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

NM_030662 MAP2K2 MAP2K2 3 UCCAGGAGUUUGUCAAUAA 164

NM_001047 SRD5A1 SRD5A1-1 GCAGAUACUUGAGCCAUUG 165

NM_002746 MAPK3 MAPK3 3 GAAACUACCUACAGUCUCU 166

NM_001047 SRD5A1 SRD5A1 4 CCGGAAAUUUGAAGAGUAU 167

NM_005178 Bcl3 Bcl3 1 GAACACCGAGUGCCAAGAA .168

NM_000348 SRD5A2 SRD5A2 3 GGACAUUUGUGUACUCACU 169

12 C

NM_002755 MAP2K1 MAP2K1 1 GCACAUGGAUGGAGGUUCU 170

NM_002755 MAP2K1 MAP2K1 2 GCAGAGAGAGCAGAUUUGA 171

NM_002755 MAP2K1 MAP2K1 3 GAGGUUCUCUGGAUCAAGU 172

NM_002755 MAP2K1 MAP2K1 4 GAGCAGAUUUGAAGCAACU 173

NM_030662 MAP2K2 MAP2K2 1 CAAAGACGAUGACUUCGAA 174

NM_030662 MAP2K2 MAP2K2 2 GAUCAGCAUUUGCAUGGAA 175

NM_030662 MAP2K2 MAP2K2 3 UCCAGGAGUUUGUCAAUAA 176

NM_030662 MAP2K2 MAP2K2 4 GGAAGCUGAUCCACCUUGA 177

12 d

NM_030662 MAP2K2 MAP2K2 3 UCCAGGAGUUUGUCAAUAA 178

NM_001047 SRD5A1 SRD5A1 1 GCAGAUACUUGAGCCAUUG 179

NM_001047 SRD5A1 SRD5A1 3 CCGGAAAUUUGAAGAGUAU 180

NM_000348 SRD5A2 SRD5A2 3 GGACAUUUGUGUACUCACU 181

13A

NM_005990 STK10     GAAACGAGAUUCCUUCAUC 182

AY406545 MADH6     CAAGAUCGGUUUUGGCAUA 183

NM_170679 SKP1A     CAAACAAUCUGUGACUAUU 184

NM_002257 KLK1     CAACUUGUUUGACGACGAA 185

NM_000942 PPIB     GAAAGGAUUUGGCUACAAA 186

NM_005083 U2AF1L1     GAGCAUGUUUACAACGUUU 187

NM_000942 PPIB     GGAAAGACUGUUCCAAAAA 188

NM_006622 SNK     ACAUUUACAUUCUCUUGGA 189

NM_000942 PPIB     GAAAGAGCAUCUACGGUGA 190

NM_005379 MYO1A     ACAAGGAGAUUUAUACCUA 191

NM_002620 PF4V1     AGGAACAUUUGGAGAGUUA 192

NM_005627 Sgk1     CAUCGUUUAUAGAGACUUA 193

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

NMA_022550 XRCC4     GAAAGUAAGCAGAAUCUAU 194

AY313906 SARS SEP     AACCAACGGUUUACGUCUA 195

NM_181523 PIK3R1     GAAAGACAAGAGACCAAUA 196

NM_020183 TRNA2     CAACAGCGAUUUUAGGAUA 197

NM_018131 C10ORF3     GGAAACAGCUGCUCAUUCA 198

NM_139025 ADAMTS13     ACAUUUGGCUGUGAUGGUA 199

NM_005767 P2RY5     GAAACUACAACUUACAUGA 200

NM_147199 MRGX1     GAUGAUGUUUUCCUACUUU 201

NM_001892 CSNK1A1     AGAAUUUGCGAUGUACUUA 202

NM_006930 SKP1A     AGGUUUGCUUGAUGUUACA 203

M15077 PPYLUC     CGAAAGGUCUUACCGGAAA 204

NM_006257 PRKCQ     CAAAGAGUAUGUCGAAUCA 205

NM_018131 C10ORF3     AAGGAAAGCUGACUGAUAA 206

NM_013391 DMGDH     CAUCAAAGCUGCCAUGGAA 207

BC025733 FADD     CAGCAUUUAACGUCAUAUG 208

NM_005541 INPP5D     AWGCUUUUACACUUACAG 209

NM_006395 GSA7     GAUCAAAGGUUUUCACUAA 210

AC146999 Human Herpes-virus 5     CAAACCAGCGCGCUAAUGA 211

NM_153202 ADAM33     CAAACAGCGUCUCCUGGAA 212

NM_005508 CCR4     GAAAGCAUAUACAGCAAUU 213

NM_002605 PDE8A     CAAAGAAGAUAACCAAUGU 214

NM_000455 STK11     GAAACAUCCUCCGGCUGAA 215

AF493910 RALA     GAGCAGAUUUUAAGAGUAA 216

NM_012184 FOXD4L1     GGACAAUUUUGCAGCAACA 217

NM_001273 CHD4     CAAAGGUGCUGCUGAUGUA 218

NM_002434 MPG     ACAUCAUUUACGGCAUGUA 219

13B

NM_004429 EFNB1     CCACACCGCUGGCCAAGAA 220

NM_002717 PPP2R2A     UAUCAAGCCUGCCAAUAUG 221

XM_110671 M11     UCAAUAAGCCAUCUUCUAA 222

NM_001282 AP2B1     GAGCUAAUCUGCCACAUUG 223

NM_001846 COL4A2     CGAAGGCGGUGGCCAAUCA 224

AF100153 CNK     GCACAUCCGUUGGCCAUCA 225

NM_001136 AGER     GCCAGGCAAUGAACAGGAA 226

NM_007122 USF1     GGAAGCCAGCGCUCAAUUG 227

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

NM_001136 AGER     GCGAGCCACUGGUGCUGAA 228

NM_018653 GPRC5C     CCACCUCCGUUGCCAUAUG 229

NM_001431 EPB41L2     GAAGGACUCUAGCCAGUUA 230

NM_000119 EPB42     GACCACACCUUGCCAUCAA 231

NM_004448 ERBB2     GCAGUUACCAGUGCCAAUA 232

NM_005971 FXYD3     GGACGCCAAUGACCUAGAA 233

NM_003494 DYSF     GAACUAUGCUGCCAUGAAG 2. 3. 4

NM_013391 DMGDH     CAUCAAAGCUGCCAUGGAA 235

NM_022353 OSGEPL1     AGACAUUGCUGCCACAGUA 236

NM_003367 USF2     GGCCAGUUCUACGUCAUGA 237

NM_172390 NFATc1     GCCAGGAGCUGAACAUUAA 238

13C

NM_005378 MYCN     CACGUCCGCUCAAGAGUGU 239

NM_000147 FUCA1     UAACAAUGCUGGGAAUUCA 240

NM_003566 EEA1     AGACAGAGCUUGAGAAUAA 241

NM_004707 APG12L     UGUUGCAGCUUCCUACUUC 242

NM_003918 GYG2     GACCAAGGCUUACUGAAUA 243

NM_004462 FDFT1     CAUAGUUGGUGAAGACAUA 244

XM_291277 SgK223     GAGCUCCACUUCAAUGAGA 245

NM_004573 Beta 2 PLC     GAACAGAAGUUACGUUGUC 246

NM_003955 SOCS3     CACCUGGACUCCUAUGAGA 247

NM_203330 CD59     CUACAACUGUCCUAACCCA 248

NM_002377 MAS1     CUACACAAUUGUCACAUUA 249

NM_153326 AKR1A1     UGAGGAGGCUGAGUAAUUC 250

NM_001749 CAPNS1     CCACAGAACUCAUGAACAU 251

NM_016735 LIMK1     UCAACUUCAUCACUGAGUA 252

NM_002393 MDM4     CGUCAGAGCUUCUCCGUAA 253

NM_021969 NR0B2     CGUAGCCGCUGCCUAUGUA 254

NM_002741 PRKCL1     ACAGCGACGUGUUCUCUGA 255

NM_014452 TNFRSF21     CAGAAGGCCUCGAAUCUCA 256

NM_139343 BIN1     GCUCAAGGCUGGUGAUGUG 257

NM_001003945 ALAD     GAUGACAUACAGCCUAUCA 258

NM_013315 TPTE     UUUAUUCGAUUCCUCGUUA 259

NM_024560 FLJ21963     UCGAGUGGAUGAUGUAAUA 260

L07868 ERBB4     AGGAUCUGCAUAGAGUCUU 261

NM_001003809 DLGAP1     CAACCUGGAUGGUGACAUG 262

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

NM_005232 EPHA1     UGAAGAACGGUACCAGAUG 263

NM_003818 CDS2     GUGAGACAGUGACGGAUUA 264

NM_153675 FOXA2     ACGAACAGGUGAUGCACUA 265

XM_496495 GGT2     AAUAAUGAAUGGACGACUU 266

NM_020676 ABHD6     GAUGACCUGUCCAUAGAUG 267

NM_000487 ARSA     UCUAUGACCUGUCCAAGGA 268

AF348074 NAT2     AUACAGAUCUGGUCGAGUU 269

U02388 CYP4F2     CAUAUUGACUUCCUGUAUU 270

14 A-14 I

EGFP     GCAAAGACCCCAACGAGAA 271

NM_012154 eIF2C2     GCACGGAAGUCCAUCUGAA 272

GCAGGACAAAGAUGUAUUA 273

GGGUCUGUGGUGAUAAAUA 274

GUAUGAGAACCCAAUGUCA 275

NM_012154 eIF2C2     GCACGGAAGUCCAUCUGAA 276

GCAGGACAAAGAUGUAUUA 277

GGGUCUGUGGUGAUAAAUA 278

GUAUGAGAACCCAAUGUCA 279

14J

NM_006218 PI3K Ca PI3K Ca-1 AUGUUUACUACCAAAUGGA 280

NM_001047 SRD5A1 SRD5A1 2 UAACUGCAGCCAACUAUUU 281

NM_030662 MAP2K2 MAP2K2 3 UCCAGGAGUUUGUCAAUAA 282

NM_001047 SRD5A1 SRD5A1-1 GCAGAUACUUGAGCCAUUG 283

NN_001047 SRD5A1 SRD5A1 4 CCGGAAAUUUGAAGAGUAU 284

14K

NM_006218 PI3K Ca PI3K Ca 1 AUGUUUACUACCAAAUGGA 285

NM_001047 SRD5A1 SRD5A1 2 UAACUGCAGCCAACUAUUU 286

NM_030662 MAP2K2 MAP2K2 3 UCCAGGAGUUUGUCAAUAA 287

NM_001047 SRD5A1 SRD5A1 1 GCAGAUACUUGAGCCAUUG 288

NM_001047 SRD5A1 SRD5A1 4 CCGGAAAUUUGAAGAGUAU 289

NM_000348 SRD5A2 SRD5A2 3 GGACAUUUGUGUACUCACU 290

M15077 PPYLUC     UGUUUGUGGACGAAGUACC 291

BC020308 GAPDH     CCUGGCCAAGGUCAUCCAU 292

NM_000942 PPIB     GAGAAAGGAUUUGGCUACA 293

Table I

Sequences used for the data in Figures 12-14

FIG.

Access No. Gene Name Denomination ARNip Diana sequence I KNOW THAT. ID NO.

14L

NM_001047 SRD5A1 SRD5A1 2 UAACUGCAGCCAACUAUUU 294

NM_030662 MAP2K2 MAP2K2 3 UCCAGGAGUUUGUCAAUAA 295

NM_001047 SRD5A1 SRD5A1 1 GCAGAUACUUGAGCCAUUG 296

NM_001047 SRD5A1 SRD5A1 4 CCGGAAAUUUGAAGAGUAU 297

NM_000348 SRD5A2 SRD5A2 3 GGACAUUUGUGUACUCACU 298

NM_001273 CHD4     CAAAGGUGCUGCUGAUGUA 299

NM_002605 PDE8A     CAAAGAAGAUAACCAAUGU 300

NM_000455 STK11     GAAACAUCCUCCGGCUGAA 301

Note: The sequences for 14L are also present in a modified form in which positions 1 and 2 of the effector and antisense strands contain 2'-O-methyl groups and position 1 of the antisense strand also contains a phosphate group in the 5 'carbon.

The above data support the hypothesis that siRNA can induce toxicity in a sequence-independent mechanism specific to the target. The authors of the present invention conducted two separate experiments to test the dependence of siRNA-induced toxicity on the mechanism of RNAi. The results of these experiments are presented below and demonstrate that toxicity is mediated by the RNAi pathway. Since previous experiments demonstrated that toxicity was not associated with the reduction of target expression, a third experiment was conducted to determine whether the chemical pattern of the modifications described in the first embodiment could eliminate toxicity.

In the first experiment, the ability of the siRNA containing a toxic motive to induce cell death was investigated in circumstances in which the RNAi mechanism was severely compromised. Previous studies revealed that eIF2C2 / hAgo2 is responsible for the cleavage of mRNA and that reducing the expression of this gene product severely disables this route. The authors of the present invention confirmed this discovery (Figure 14 a-i) and then evaluated the importance of this route in newly discovered siRNA-induced toxicity. To test this, cells transfected with the cluster (T1) of eIF2C2 / hAgo2 siRNA were subsequently transfected with toxic siRNA containing AAA / UUU or GCCA / UGG motifs (Figure 14a, "Experiment"). The results of these experiments demonstrated that in the absence of an intact RNAi pathway, toxic siRNAs were unable to induce the cell death phenotype (Figure 14j). Since parallel experiments in which the RNAi pathway was left intact exhibited the characteristic toxicity of these sequences, it was concluded that an intact RNAi pathway was necessary for siRNA-induced toxicity. This experiment strongly supports the hypothesis that the toxic siRNA induces its phenotype through the RNAi pathway. Since the toxicity observed is not related to the level or degree of reduction of target expression, it is likely that nonspecificity is responsible for the toxic phenotype observed.

Additional support for the involvement of the RNAi pathway in the toxicity of siRNA came from an experiment in which the size of the duplex was reduced from 19 bp to 17 bp. Previous studies have shown that duplexes that are shorter than 19 bp sequences are directed to mRNA sequences inefficiently, most likely due to the fact that Dicer and / or RISC fail to mediate RNAi when the length of the sequence duplex falls below 19bp (Elbashir, SM et al. "Duplexes of 21-nucleotide RNAs through RNA interference in cultured mammalian cells" Nature 2001 May 24; 411 (6836): 494-8). When the length of the toxic 19-bp siRNA, known was reduced by 2 bp (17 bp in total length, without interruption of the motif) the level of toxicity was drastically reduced (Figure 14k), suggesting that entry is necessary and / or the processing by RISC for the induction of toxicity. These results again imply the route of the RNAi in this form of cellular toxicity induced by siRNA. Since the toxicity observed is not related to the level or degree of reduction in the expression of the target, it is likely that nonspecificity is responsible for the toxic phenotype observed.

As noted above, it has been shown that the modifications described in the first embodiment of the invention eliminate nonspecific effects. Since siRNA-induced cellular toxicity is dependent on

RNAi, but is not related to the reduction of target expression, the authors of the present invention decided to test whether modifications that eliminate nonspecificity nullify cellular toxicity induced by siRNA. To achieve this, a variation of the chemical modification pattern described in the first embodiment was added to the siRNAs that are known to induce toxicity in an RNAi-dependent mechanism. 5 Specifically, siRNA was synthesized to carry the following modifications: 2'-O-methyl groups at positions 1 and 2 of both the effector and antisense strands, plus a 5 'phosphate group at carbon 5 of the 5' terminal antisense nucleotide . As shown in Figure 14-1, when eight separate, unmodified toxic siRNAs (MAP2K2 d3, SRD5A1 d1, d2, SRD5A1 SRD5A1 D4, D3, SRD5A2 PDE8A, STK11 and CHD4) were transfected into cells, each reduced cell viability below 75%. On the contrary, the chemical modification of

10 eight duplexes markedly reduced the toxicity induced by siRNA without significantly altering the reduction of the specific expression of the target. These results strongly support the premise that the siRNA-induced toxicity of siRNA containing AAA / UUU or AMCC / UGGC is the result of nonspecific effects. More importantly, the results presented here suggest that nonspecific induced phenotypes can be eliminated by adding the modifications of the invention.

These studies have been extended by evaluating the effects of the modification patterns of the invention on thirty-seven additional siRNAs that target luciferase (Accession No. M15077). Specifically, the sequences listed in Table II below were synthesized in both the modified (2'-O-methyl at positions 1 and 2 of the effector strand, 2'-O-methyl at position 2 of the strand

20 antisense, 5 'carbon phosphate of the 5' terminal nucleotide of the antisense strand) and as unmodified, and were transfected into HeLa cells (5,000 cells per well, 10 µM siRNA, 0.1 microliters of Lipofectamine 2000 per well) . Subsequently, the level of cell death in each culture was measured 72 hours after transfection using Alamar Blue.

25 Table II lists the toxic luc sequences used in this study and includes (1) the SEC. ID NO. (right column) and (2) the sequence (left column, 5 '→ 3', effector thread).

TABLE II

Paseo with luc - Toxic Sequences

SEQUENCE

SEC. ID NO.

AGAUCCUCAUAAAGGCCAA

302

AGAGAUCCUCAUAAAGGCC

303

AAUCAGAGAGAUCCUCAUA

304

AAAAUCAGAGAGAUCCUCA

305

GAAAAAUCAGAGAGAUCCU

306

GCAAGAAAAAUCAGAGAGA

307

CUCGACGCAAGAAAAAUCA

309

GGAAAACUCGACGCAAGAA

309

CUUACCGGAAAACUCGACG

310

GUCUUACCGGAAAACUCGA

311

AGGUCUUACCGGAAAACUC

312

CGAAAGGUCUUACCGGAAA

313

AAGUACCGAAAGGUCUUAC

314

UGGACGAAGUACCGAAAGG

315

UGUUUGUGGACGAAGUACC

316

UGUGUUUGUGGACGAAGUA

317

GUUGUGUUUGUGGACGAAG

318

UUGCGCGGAGGAGUUGUGU

319

AAAGUUGCGCGGAGGAGUU

320

AGUCAAGUAACAACCGCGA

321

TABLE II

Paseo with luc - Toxic Sequences

SEQUENCE

SEC. ID NO.

GUCGCCAGUCAAGUAACAA

322

GAUUACGUCGCCAGUCAAG

323

UGGAUUACGUCGCCAGUCA

324

CGUGGAUUACGUCGCCAGU

325

AGAUCGUGGAUUACGUCGC

326

AGAGAUCGUGGAUUACGUC

327

AAAAAGAGAUCGUGGAUUA

328

ACGGAAAAAGAGAUCGUGG

329

UGACGGAAAAAGAGAUCGU

330

GAGCACGGAAAGACGAUGA

331

UGGAGCACGGAAAGACGAU

332

UUUGGAGCACGGAAAGACG

333

GUUUUGGAGCACGGAAAGA

334

UGUUGUUUUGGAGCACGGA

335

CCGUUGUUGUUUUGGAGCA

336

AACUUCCCGCCGCCGUUGU

337

UGAACUUCCCGCCGCCGUU

338

The results of these studies are presented in Figure 14m and show (1) in twenty-five of the thirty-seven cases, the level of toxicity was significantly reduced after the addition of the chemical modifications of the invention, and (2) in twenty-two of In the twenty cases, the level of toxicity fell below the 25% level previously established as the line of discrimination between toxic and non-toxic transfections. Since additional studies designed to compare the functionality of modified and unmodified luc sequences showed comparable activity in both groups of molecules (Figure 14n, HEK 293 cells, 20,000 cells per well, 0.3 microliters of Lipofectamine 2000 per well, 72 hour time point, 100 nM siRNA), these findings strongly support the premise that (1) siRNA-induced toxicity caused by siRNA that

10 contains AAA / UUU or AMCC / UGGC is the result of nonspecific effects, and (2) that nonspecific induced phenotypes can be greatly reduced or eliminated by the addition of the modifications of the invention.

Example 11

15 Synergistic effects of the chemical modifications of the invention and the grouping on elimination of nonspecific effects

To evaluate the value of the combination of the chemical modifications of the invention and the grouping to eliminate

20 nonspecific silencing, four siRNAs were tested as well as the respective clustering of the molecules, targeting human cyclophilin B (PPIB) to determine the nonspecific effects (in both modified and unmodified forms) using gene expression based profiling in microarrays To achieve this, the sequences directed to PPIB (see below) were first synthesized in the unmodified and modified forms (2'-O-methyl at positions 1 and 2 of the effector strand, 2'-O-methyl in the position 2 of the strand

25 antisense, 5 'carbon phosphate of the 5' nucleotide of the antisense strand).

Sequences: effector strand

PPIB-1 / Cl: 5'-GAAAGAGCAUCUACGGUGA-3 '(SEQ ID NO. 339) PPIB -2 / C2: 5'-GAAAGGAUUUGGCUACAAA-3' (SEQ ID NO. 340) PPIB -3 / C3: 5 ' -ACAGCAAAUUCCAUCGUGU-3 '(SEQ ID NO. 341) PPIB -4 / C4: 5'-GGAAAGACUGUUCCAAAAA-3' (SEQ ID NO. 342)

Subsequently, the duplexes were transfected into human HeLa cells (10K cells per well of a 96-well plate, 0.4 µL of Dharmafect 1 lipid per well (Dharmacon, Inc.), concentration of 100 nM. Note, in the case of the grouping, the concentration of each duplex was 25 nM, therefore the total concentration remained constant with studies on individual siRNAs). Cell lysate products were then collected for microarray analysis (24 hours after transfection) and purification of total RNA was performed using RNeasy columns of Qiagen with digestion with DNase in column. RNA integrity was analyzed with the 6000 Nano LabChip RNA in Agilent Bioanalyzer 2100. To carry out the gene expression profiles, the following procedures were carried out: for each sample, 650 ng of total RNA was amplified and labeled with Cy3 or Cy5 (Perkin Elmer) using Agilent Low Input RNA Fluorescent Linear Amplification Kit. Hybridizations were performed using Human 1A (V2) Oligo Microarrays of Agilent (http://www.agilent.com). The hybridization reference (Cy3) were simulated transfected cells or non-transfected cells, as indicated. The slides were washed and dried using 6X and 0.06X SSPE with 0.025% N-lauroylsarcosine, pure acetonitrile, and Agilent proprietary non-aqueous drying and stabilization solution. Subsequently, they were scanned in an Agilent Microarray Scanner (model G2505B) and the raw image was processed using Feature Extraction (v7.1.1.). Additional analysis / data processing was performed using Spotfire Decision Site 8.0 and Spotfire Functional Genomics Module. The low signal genes were removed from the analysis by applying a Log cut (additive signal of red and green in pixels)> 2.8 from the data of a representative auto-self matrix. No elimination of outliers was carried out. A double cut-off point (Log Ratio> 0.3 or <-0.3) was applied to the genes used in the comparative analysis.

The results of the siRNA analysis directed to cyclophilin B showed that although the level of reduction of the expression of the gene chosen as the target remained constant in the modified and unmodified duplexes (data not shown), in all cases the addition of the pattern Chemical modification of the invention reduced the number of non-specific genes (downregulated more than twice) by 50% or more. For duplexes C1, C2, C3, and C4, the number of nonspecific targets was reduced from 5 → 2, 57 → 23, 12 → 6, and 72 → 21, respectively (Note, in the case of C2, it is believed 17 of the 23 nonspecific targets are experimental artifacts, therefore the reduction in this case can be as large as 57 → 6). These results support the above results (see Figures 7-11) which show that the addition of the modifications of the invention strongly suppress the nonspecific effects. Figure 15c shows that the number of nonspecific targets generated by the unmodified cluster is less than that predicted from the four individual siRNAs (i.e., the sum of the nonspecific targets generated by each duplex = 5 + 57 + 12 + 72 = 146 vs. the grouping of the four duplexes = 65). Therefore, in this case, grouping by itself can reduce the number of nonspecific targets by approximately 55%. To the surprise of the authors of the present invention, only four nonspecific targets were identified using modified clusters. Compared to the total number of nonspecific targets observed in individual, unmodified siRNAs (146), this represents a 97.2% reduction in the total number of nonspecific targets. Compared to the number of nonspecific targets observed in the unmodified groupings (65), this change (65 → 4) represents a net reduction of 94%. These unexpected and surprising results demonstrate that the combination of the modifications of the invention with the clusters provides a surprising drastic advantage over the individual siRNAs or the siRNA pools.

To determine whether the observed benefits of combining chemical modifications with clustering are limited to cyclophilin B-directed sequences, siRNAs targeting a second gene (human MEK1, MAP2K1, Accession No. NM_002755) were subjected to a format identical. The sequences used in this study are shown below:

MAP2K1-1 (M1): 5'-GCACAUGGAUGGAGGUUCU-3 '(SEQ. ID NO. 343)

MAP2K1-2 (M2): S'-GCAGAGAGAGCAGAUUUGA-3 '(SEQ. ID NO. 344)

MAP2K1-4 (M4): 5'-GAGCAGAUUUGAAGCAACU-3 '(SEQ. ID NO. 345)

MAP2K1-5 (M5): 5'-CCAGAAAGCUAAUUCAUCU-3 '(SEQ. ID NO. 346)

As was the case with cyclophilin B, the addition of the chemical modification of the invention to the siRNA directed to MAP2K1 does not appreciably affect the reduction of the expression of the gene chosen as the target. But as previously noted, the addition of the chemical modifications of the invention to the individual siRNA directed to MAP2K1 reduced (in all cases with the exception of one suspected of being an artifact, ie M4) the number of genes that they were unspecific targets twice (or more) in 50% or more (85 → 8, 9 → 2, 58 → 32, 15 → 41 (artifact), see Figures 15d-f). In addition, as observed in previous studies with PPIB, the grouping of

Individual duplexes generated fewer nonspecific targets than the combined number observed with individual siRNAs (clustered nonspecific targets = 26 vs. combined nonspecific nonspecific targets = 167). While this represents an 85% reduction in the total number of nonspecific targets, the combination of chemical modifications with the cluster again increased the specificity of the cluster alone. It was observed that only two genes were downregulated twice or more when clusters containing siRNA directed to modified MAP2K1 were used. In comparison with the results generated with unmodified, individual siRNAs (167) this represents a 98.8% reduction in the number of nonspecific targets. Compared to unmodified clusters (26 unspecific targets in total), the number of nonspecific targets observed in the modified clusters (2) represents a 92.3% reduction in the total number of nonspecific genes. In

10 together, these data demonstrate that the combination of the modifications of the invention and the grouping provided a level not previously obtainable of specificity in the reduction of gene expression. This increase in specificity is observed independently of the gene chosen as the target and could greatly reduce the number of false positives p. ex. in high-performance scrutiny designed to identify potential drug targets.

SEQUENCE LIST

<110> DHARMACON, INC. LEAKE, Devin YURIY, Fedorov REYNOLDS, Angela KHVOROVA, Anastasia MARSHALL, William

<120> Modified Polynucleotides to reduce the Effects of Inespecificity on RNA Interference

<130> 13636PCT

<140> Pending assignment

<141> 2005-03-31

<150>

 60/630228 <151> 2004-11-22

<150>

 11/019831 <151> 2004-12-22

<150>

 PCT / US04 / 10343

<151> 2004-04-01

<160> 346

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 1 ugcugaccuc uguuaccuc 19

<210> 2

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 2 gacaugcgaa ugacacuag 19

<210> 3

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 3 ccuacagaga acugcgguu 19

<210> 4

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 4 agcugaccuc uguuaccuc

19

<210> 5 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 5 ccuacagaga acugcgguu

 19

<210> 6 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 6 gucaucagcu uugugccac

19

<210> 7 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 7 gacaugcgaa ugacacuag

19

<210> 8 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 8 agaggaacuc ucugcaagc

19

<210> 9 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 9 uggaggggaa ugcucagaa

19

<210> 10 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 10 uaaagauggc acuuucccg

19

<210> 11 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 11 ugcugaccuc uguuaccuc

19

<210> 12 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 12 gcucacgguc auuaccgag

 19

<210> 13 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 13 ccuacagaga acugcgguu

19

<210> 14 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 14 ggcuuccuua caaggagau

19

<210> 15 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 15 ccuacagaga acugcgguu

19

<210> 16 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 16 cgggucuugu guccuucaa

19

<210> 17 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> RNAip effector sequence

<400> 17 gucaucagcu uugugccac

19

<210> 18 <211> 4 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip loop sequences

<400> 18 uucg

4

<210> 19 <211> 9 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip loop sequences

<400> 19 uuuguguag

9

<210> 20 <211> 10 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip loop sequences

<400> 20 cuuccuguca

10

<210> 21 <211> 7 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip loop sequences

<400> 21 auaugug <210> 22 <211> 21 <212> RNA <213> Artificial Sequence

7

<220>

<223> effector strand of siRNA

<400> 22 gugauguaug ucagagaguu u

twenty-one

<210> 23 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 23 gaaaaaucag agagauccu

19

<210> 24 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 24 uaccggaaaa cucgacgca

19

<210> 25 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 25 acgucgccag ucaaguaac

19

<210> 26 <211> 19

<212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 26 gauuacgucg ccagucaag

19

<210> 27 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA <400> 27 agagaucgug gauuacguc

19

<210> 28 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 28 uguuguuuug gagcacgga

19

<210> 29 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 29 ccuacagaga acugcgguu

19

<210> 30 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 30 ggcuuccuua caaggagau

19

<210> 31 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 31 cgggucuugu guccuucaa

19

<210> 32 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 32

acgggcaagg ccaaguggg

19

<210> 33 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence <400> 33

cgggcaaggc caaguggga

19

<210> 34 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 34 gggcaaggcc aagugggau

19

<210> 35 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 35 ggcaaggcca agugggaug

<210> 36 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 36

gcaaggccaa gugggaugc

19

<210> 37 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 37 caaggccaag ugggaugcc

19

<210> 38 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 38 aaggccaagu gggaugccu

19

<210> 39 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 39 aggccaagug ggaugccug

19

<210> 40 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 40 ggccaagugg gaugccugg

19

<210> 41 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 41 gccaaguggg augccugga

19

<210> 42 <211> 19

<212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 42 ccaaguggga ugccuggaa

19

<210> 43 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 43 caagugggau gccuggaau

19

<210> 44 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 44 aagugggaug ccuggaaug

19

<210> 45 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 45 agugggaugc cuggaauga

19

<210> 46 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 46 gugggaugcc uggaaugag

19

<210> 47 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 47 ugggaugccu ggaaugagc

19

<210> 48 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 48 gggaugccug gaaugagcu

19

<210> 49 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 49 ggaugccugg aaugagcug

19

<210> 50 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 50 raugccugga augagcuga

<210> 51 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 51 augccuggaa ugagcugaa

19

<210> 52 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 52 ugccuggaau gagcugaaa

19

<210> 53 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 53 gccuggaaug agcugaaag

19

<210> 54 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 54 ccuggaauga gcugaaagg

19

<210> 55 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 55 cuggaaugag cugaaaggg

 19

<210> 56 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 56 uggaaugagc ugaaaggga

19

<210> 57 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 57 ggaaugagcu gaaagggac

19

<210> 58 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 58 gaaugagcug aaagggacu

19

<210> 59 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 59 aaugagcuga aagggacuu

19

<210> 60 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 60 augagcugaa agggacuuc

19

<210> 61 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 61 ugagcugaaa gggacuucc

19

<210> 62 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 62 gagcugaaag ggacuucca

19

<210> 63 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 63 agcugaaagg gacuuccaa

 19

<210> 64 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 64 gcugaaaggg acuuccaag

19

<210> 65 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 65 cugaaaggga cuuccaagg

19

<210> 66 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 66 ugaaagggac uuccaagga

19

<210> 67 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 67 gaaagggacu uccaaggaa

19

<210> 68 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 68 aaagggacuu ccaaggaag

19

<210> 69

<211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 69 aagggacuuc caaggaaga

19

<210> 70 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 70 agggacuucc aaggaagau

19

<210> 71 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 71 gggacuucca aggaagaug

19

<210> 72 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 72 ugacuuccaa ggaagaugc

19

<210> 73 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 73 gacuuccaag gaagaugcc

19

<210> 74 <211> 19 <212> RNA <213> Artificial Sequence

<220> <223> siRNA target sequence

<400> 74 acuuccaagg aagaugcca

19

<210> 75 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 75 cuuccaagga agaugccau

19

<210> 76 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 76 uuccaaggaa gaugccaug

19

<210> 77 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 77 uccaaggaag augccauga

19

<210> 78 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 78 ccaaggaaga ugccaugaa

 19

<210> 79 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 79

caaggaagau gccaugaaa <210> 80 <211> 19 <212> RNA <213> Artificial Sequence

     19

<220>

<223> siRNA target sequence

<400> 80 aaggaagaug ccaugaaag

19

<210> 81 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 81 aggaagaugc caugaaagc

19

<210> 82 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 82 ggaagaugcc augaaagcu

19

<210> 83 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 83 gaagaugcca ugaaagcuu

19

<210> 84 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 84 aagaugccau gaaagcuua

19

<210> 85 <211> 19 <212> RNA

<213> Artificial Sequence <220>

<223> siRNA target sequence

<400> 85 agaugccaug aaagcuuac

19

<210> 86 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 86 gaugccauga aagcuuaca

 19

<210> 87 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 87 augccaugaa agcuuacau

19

<210> 88 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 88 ugccaugaaa gcuuacauc

19

<210> 89 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 89 gccaugaaag cuuacauca

19

<210> 90 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 90 ccaugaaagc uuacaucaa

19

<210> 91 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 91 caugaaagcu uacaucaac

19

<210> 92 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 92 augaaagcuu acaucaaca

19

<210> 93 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 93 ugaaagcuua caucaacaa

19

<210> 94 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 94 gaaagcuuac aucaacaaa

 19

<210> 95 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 95 aaagcuuaca ucaacaaag

19

<210> 96 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 96 aagcuuacau caacaaagu

19

<210> 97 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 97 agcuuacauc aacaaagua

19

<210> 98 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 98 gcuuacauca acaaaguag

19

<210> 99 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 99 cuuacaucaa caaaguaga

19

<210> 100 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 100 uuacaucaac aaaguagaa

19

<210> 101 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 101 uacaucaaca aaguagaag

19

<210> 102 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 102 acaucaacaa aguagaaga

 19

<210> 103 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 103 caucaacaaa guagaagag

19

<210> 104 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 104 aucaacaaag uagaagagc

19

<210> 105 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 105 ucaacaaagu agaagagcu

19

<210> 106 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 106 caacaaagua gaagagcua

19

<210> 107 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 107 aacaaaguag aagagcuaa

19

<210> 108 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 108 acaaaguaga agagcuaaa

19

<210> 109 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 109 caaaguagaa gagcuaaag

19

<210> 110 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 110 aaaguagaag agcuaaaga

19

<210> 111 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 111 aaguagaaga gcuaaagaa

19

<210> 112 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 112 aguagaagag cuaaagaaa

19

<210> 113 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 113 guagaagagc uaaagaaaa

19

<210> 114 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 114 uagaagagcu aaagaaaaa

19

<210> 115 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 115 agaagagcua aagaaaaaa

19

<210> 116 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 116 gaagagcuaa agaaaaaau

19

<210> 117 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 117 aagagcuaaa gaaaaaaua

19

<210> 118 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 118 agagcuaaag aaaaaauac

19

<210> 119 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 119 gagcuaaaga aaaaauacg

 19

<210> 120 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 120 agcuaaagaa aaaauacgg

19

<210> 121 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 121 gcuaaagaaa aaauacggg

19

<210> 122 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 122 gaaguacauc cauuauaag

19

<210> 123 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 123 aaacagaucu uuacaagcu

19

<210> 124 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 124 uuucaagugu cuccuaaua

19

<210> 125 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 125 gcaaagaaca ucauccaua

19

<210> 126 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 126 gcagagagag cagauuuga

19

<210> 127 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 127 ucaaggaacu cgaucguau

19

<210> 128 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 128 gacaugaaau ccaacaaua

19

<210> 129 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 129

ucaagugucu ccuaauaug

19

<210> 130 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 130 caaagacgau gacuucgaa

19

<210> 131 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 131 ggaagcugau ccaccuuga

19

<210> 132 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 132 caagaggauu gaaguagaa

19

<210> 133 <211> 19 <212> RNA

<213> Artificial Sequence <220>

<223> siRNA target sequence

<400> 133 ccaaagcucu ggacuuauu

<210> 134 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 134 guacgacaac cgggagaua

19

<210> 135 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 135 agauagugau gaaguacau

19

<210> 136 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 136 gggagauagu gaugaagua

19

<210> 137 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 137 gaaaugauug cacuauuga

19

<210> 138 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 138 gcacggagau guugcagua

19

<210> 139 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 139 gcuacuaucu gauuuacug

19

<210> 140 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 140 caagggaggu uacaccaaa

19

<210> 141 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 141 gagguucucu ggaucaagu

19

<210> 142 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 142 gcuacacgca guugcagua

19

<210> 143 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 143 agacugaccu guacaaguu

19

<210> 144 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 144 gaucagcauu ugcauggaa

19

<210> 145 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 145 ggaacucgau cguaucauu

19

<210> 146 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 146 cgacaagacu gccgagaga

19

<210> 147 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 147 gagccuuacu gccuuugua

19

<210> 148 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 148 cugaagaaag cauugacua

19

<210> 149 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 149 ggccggaggc gcuuuacua

19

<210> 150 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 150 guacagggcu ccagaaauu

19

<210> 151 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 151 ucgacgcagu ggacauuaa

19

<210> 152 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 152 aacuagaagu auguugcua

19

<210> 153 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 153 gaccggaugu uaaccuuua

19

<210> 154 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 154 uugggugucu ucuuauuua

19

<210> 155 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 155 aauggcuuug aaucuuugg

19

<210> 156 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> SIARN target sequence

<400> 156 gcacauggau ggagguucu

19

<210> 157 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 157 ggauucaguu ggagugauu

19

<210> 158 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 158 caaagaacau cauccauag

19

<210> 159 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 159 gaaagccuau gccacuguu

19

<210> 160 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 160 gcuaugcccu ggccacuug

19

<210> 161 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 161 gagcagauuu gaagcaacu

19

<210> 162 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 162 uaacugcagc caacuauuu

19

<210> 163 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 163 auguuuacua ccaaaugga

19

<210> 164 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 164 uccaggaguu ugucaauaa

19

<210> 165 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 165 gcagauacuu gagccauug

19

<210> 166 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 166 gaaacuaccu acagucucu

19

<210> 167 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 167 ccggaaauuu gaagaguau

19

<210> 168 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 168 gaacaccgag ugccaagaa

19

<210> 169 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 169 ggacauuugu guacucacu

19

<210> 170 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 170 gcacauggau ggagguucu

19

<210> 171 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 171 gcagagagag cagauuuga

19

<210> 172 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 172 gagguucucu ggaucaagu

19

<210> 173 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 173 gagcagauuu gaagcaacu

19

<210> 174 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 174 caaagacgau gacuucgaa

19

<210> 175 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 175 gaucagcauu ugcauggaa

19

<210> 176 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 176 uccaggaguu ugucaauaa

19

<210> 177 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 177 ggaagcugau ccaccuuga

19

<210> 178 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 178 uccaggaguu ugucaauaa

19

<210> 179 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 179 gcagauacuu gagccauug

19

<210> 180 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 180 ccggaaauuu gaagaguau

19

<210> 181 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 181 ggacauuugu guacucacu

19

<210> 182 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 182 gaaacgagau uccuucauc

19

<210> 183 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 183 caagaucggu uuuggcaua

19

<210> 184 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 184 caaacaaucu gugacuauu

19

<210> 185 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 185 caacuuguuu gacgacgaa

19

<210> 186 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 186 gaaaggauuu ggcuacaaa

19

<210> 187 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 187 gagcauguuu acaacguuu

19

<210> 188 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 188 ggaaagacug uuccaaaaa

19

<210> 189 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 189 acauuuacau ucucuugga

19

<210> 190 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 190 gaaagagcau cuacgguga

19

<210> 191 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

400> 191 acaaggagau uuauaccua

19

<210> 192 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 192 aggaacauuu ggagaguua

19

<210> 193 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 193 caucasianguuuau agagacuua

19

<210> 194 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

400> 194 gaaaguaagc agaaucuau

19

<210> 195 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 195 aaccaacggu uuacgucua

19

<210> 196 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 196 gaaagacaag agaccaaua

19

<210> 197 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 197 caacagcgau uuuaggaua

19

<210> 198 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 198 ggaaacagcu gcucauuca

19

<210> 199 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 199 acauuuggcu gugauggua

19

<210> 200 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 200 gaaacuacaa cuuacauga

19

<210> 201 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 201 gaagaagaaa accuacuuu

19

<210> 202 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 202 agaauuugcg auguacuua

19

<210> 203 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 203 agguuugcuu gauguuaca

19

<210> 204 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 204 cgaaaggucu uaccggaaa

19

<210> 205 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 205 caaagaguau gucgaauca

19

<210> 206 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 206 aaggaaagcu gacugauaa

19

<210> 207 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 207 caucaaagcu gccauggaa

19

<210> 208 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 208 cagcauuuaa cgucauaug

19

<210> 209 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 209 auugcguuua cacuuacag

19

<210> 210 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 210 gaucaaaggu uuucacuaa

19

<210> 211 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 211 caaaccagcg cgcuaauga

19

<210> 212 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 212 caaacagcgu cuccuggaa

19

<210> 213 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 213 gaaagcauau acagcaauu

19

<210> 214 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 214 caaagaagau aaccaaugu

19

<210> 215 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 215 gaaacauccu ccggcugaa

19

<210> 216 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 216

gagcagauuu uaagaguaa

<210> 217 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 217 ggacaauuuu gcagcaaca

19

<210> 218 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

400> 218 caaaggugcu gcugaugua

19

<210> 219 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 219 acaucauuua cggcaugua

19

<210> 220 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 220 ccacaccgcu ggccaagaa

19

<210> 221 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 221 uaucaagccu gccaauaug

19

<210> 222 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 222 ucaauaagcc aucuucuaa

19

<210> 223 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 223 gagcuaaucu gccacauug

19

<210> 224 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 224 cgaaggcggu ggccaauca

19

<210> 225 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 225 gcacauccgu uggccauca

19

<210> 226 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 226 gccaggcaau gaacaggaa

19

<210> 227 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 227 ggaagccagc gcucaauug

19

<210> 228 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 228 gcgagccacu ggugcugaa

19

<210> 229 <211> 19 <212> RNA

<213> Artificial Sequence <220>

<223> siRNA target sequence

<400> 229 ccaccuccgu ugccauaug

19

<210> 230 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 230 gaaggacucu agccaguua

19

<210> 231 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 231 gaccacaccu ugccaucaa

19

<210> 232 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

400> 232 gcaguuacca gugccaaua

19

<210> 233 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 233 ggacgccaau gaccuagaa

19

<210> 234 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 234 gaacuaugcu gccaugaag

19

<210> 235 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 235 caucaaagcu gccauggaa

19

<210> 236 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 236 agacauugcu gccacagua

19

<210> 237 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 237 ggccaguucu acgucauga

19

<210> 238 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 238 gccaggagcu gaacauuaa

19

<210> 239 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> SIARN target sequence

<400> 239J

cacguccgcu caagagugu

19

<210> 240 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 240 uaacaaugcu gggaauuca

19

<210> 241 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 241 agacagagcu ugagaauaa

19

<210> 242 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 242 uguugcagcu uccuacuuc

19

<210> 243 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 243 gaccaaggcu uacugaaua

19

<210> 244 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 244 cauaguuggu gaagacaua

19

<210> 245 <211> 19 <212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 245 gagcuccacu ucaaugaga

19

<210> 246 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 246 gaacagaagu uacguuguc

19

<210> 247 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 247 caccuggacu ccuaugaga

19

<210> 248 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 248 cuacaacugu ccuaaccca

19

<210> 249 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 249 cuacacaauu gucacauua

19

<210> 250 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

400> 250 ugaggaggcu gaguaauuc

19

<210> 251 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 251 ccacagaacu caugaacau

19

<210> 252 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 252 ucaacuucau cacugagua

19

<210> 253 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 253 cgucagagcu ucuccguaa

19

<210> 254 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 254 cguagccgcu gccuaugua

19

<210> 255 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 255 acagcgacgu guucucuga

19

<210> 256 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 256 cagaaggccu cgaaucuca

19

<210> 257 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 257 gcucaaggcu ggugaugug

19

<210> 258 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 258 gaugacauac agccuauca

19

<210> 259 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 259 uuuauucgau uccucguua

19

<210> 260 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 260 ucgaguggau gauguaaua

19

<210> 261 <211> 19 <212> RNA

<213> Artificial Sequence

<220>

<223> Diagnostic analysis of siRNA for Diana Sequence of siRNA

<400> 261 aggaucugca uagagucuu

19

<210> 262 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 262 caaccuggau ggugacaug

19

<210> 263 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 263 ugaagaacgg uaccagaug

19

<210> 264 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 264 gugagacagu gacggauua

19

<210> 265 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 265 acgaacaggu gaugcacua

19

<210> 266 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 266 aauaaugaau ggacgacuu

19

<210> 267 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 267 gaugaccugu ccauagaug

19

<210> 268 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 268 ucuaugaccu guccaagga 19

<210> 269 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 269 auacagaucu ggucgaguu 19

<210> 270 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 270 cauauugacu uccuguauu 19

<210> 271 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 271 gcaaagaccc caacgagaa 19

<210> 272 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 272 gcacggaagu ccaucugaa

19

<210> 273 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 273 gcaggacaaa gauguauua

19

<210> 274 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 274 gggucugugg ugauaaaua

19

<210> 275 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 275 guaugagaac ccaauguca

19

<210> 276 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 276 gcacggaagu ccaucugaa 19

<210> 277 <211> 19 <212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 277 gcaggacaaa gauguauua 19

<210> 278

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 278 gggucugugg ugauaaaua 19

<210> 279

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 279 guaugagaac ccaauguca 19

<210> 280

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 280 auguuuacua ccaaaugga 19

<210> 281

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 281 uaacugcagc caacuauuu 19

<210> 282

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 282 uccaggaguu ugucaauaa 19

<210> 283 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 283 gcagauacuu gagccauug 19

<210> 284 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 284 ccggaaauuu gaagaguau

<210> 285 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 285 auguuuacua ccaaaugga

19

<210> 286 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 286 uaacugcagc caacuauuu

19

<210> 287 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 287 uccaggaguu ugucaauaa

19

<210> 288 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 288 gcagauacuu gagccauug

19

<210> 289 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 289 ccggaaauuu gaagaguau

19

<210> 290 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 290 ggacauuugu guacucacu

19

<210> 291 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 291 uguuugugga cgaaguacc

19

<210> 292 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 292 ccuggccaag gucauccau

19

<210> 293 <211> 19 <212> RNA

<213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 293 gagaaaggau uuggcuaca

19

<210> 294 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

400> 294 uaacugcagc caacuauuu

19

<210> 295 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 295 uccaggaguu ugucaauaa

19

<210> 296 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 296 gcagauacuu gagccauug

19

<210> 297 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 297 ccggaaauuu gaagaguau

19

<210> 298 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 298 ggacauuugu guacucacu

19

<210> 299 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 299 caaaggugcu gcugaugua

<210> 300 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 300 caaagaagau aaccaaugu

19

<210> 301 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> siRNA target sequence

<400> 301 gaaacauccu ccggcugaa

19

<210> 302 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 302 agauccucau aaaggccaa

19

<210> 303 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 303 agagauccuc auaaaggcc

19

<210> 304 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 304 aaucagagag auccucaua

19

<210> 305 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 305 aaaaucagag agauccuca

19

<210> 306 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 306 gaaaaaucag agagauccu

19

<210> 307 <211> 19 <212> RNA <213> Artificial Sequence <220>

<223> SiRNA sensestrand

<400> 307 gcaagaaaaa ucagagaga

19

<210> 308 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 308 cucgacgcaa gaaaaauca

19

<210> 309 <211> 19 <212> RNA <213> Artificial Sequence

<220> <223> effector strand of siRNA

<400> 309 ggaaaacucg acgcaagaa

19

<210> 310 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 310 cuuaccggaa aacucgacg

 19

<210> 311 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 311 gucuuaccgg aaaacucga

19

<210> 312 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 312 aggucuuacc ggaaaacuc

19

<210> 313 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 313 cgaaaggucu uaccggaaa

19

<210> 314 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 314 aaguaccgaa aggucuuac <210> 315 <211> 19 <212> RNA <213> Artificial Sequence

19

<220>

<223> ARNip effector strand

<400> 315 uggacgaagu accgaaagg

19

<210> 316 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 316 uguuugugga cgaaguacc

19

<210> 317 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 317 uguguuugug gacgaagua

19

<210> 318 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 318 guuguguuug uggacgaag

19

<210> 319 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 319 uugcgcggag gaguugugu

19

<210> 320 <211> 19

<212> RNA <213> Artificial Sequence <220>

<223> ARNip effector strand

<400> 320 aaaguugcgc ggaggaguu

19

<210> 321 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 321 agucaaguaa caaccgcga

19

<210> 322 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> effector strand of siRNA

<400> 322 gucgccaguc aaguaacaa

19

<210> 323 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 323 gauuacgucg ccagucaag

19

<210> 324 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 324 uggauuacgu cgccaguca

19

<210> 325 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 325 cguggauuac gucgccagu

19

<210> 326 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 326 agaucgugga uuacgucgc

19

<210> 327 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 327 agagaucgug gauuacguc

19

<210> 328 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 328 aaaaagagau cguggauua

19

<210> 329 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 329 acggaaaaag agaucgugg

19

<210> 330 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 330 ugacggaaaa agagaucgu

19

<210> 331 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 331 gagcacggaa agacgauga

19

<210> 332 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 332 uggagcacgg aaagacgau

19

<210> 333 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 333 uuuggagcac ggaaagacg

19

<210> 334 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 334 guuuuggagc acggaaaga

19

<210> 335 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 335 uguuguuuug gagcacgga

19

<210> 336 <211> 19 <212> RNA

<213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 336 ccguuguugu uuuggagca

19

<210> 337 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 337 aacuucccgc cgccguugu

19

<210> 338 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 338 ugaacuuccc gccgccguu

19

<210> 339 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 339 gaaagagcau cuacgguga

19

<210> 340 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 340 gaaaggauuu ggcuacaaa

19

<210> 341 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 341 acagcaaauu ccaucgugu

19

<210> 342 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 342 ggaaagacug uuccaaaaa

19

<210> 343 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 343 gcacauggau ggagguucu

19

<210> 344 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 344 gcagagagag cagauuuga

19

<210> 345 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 345 gagcagauuu gaagcaacu

19

<210> 346 <211> 19 <212> RNA <213> Artificial Sequence

<220>

<223> ARNip effector strand

<400> 346 ccagaaagcu aauucaucu

19

Claims (4)

1. A double stranded siRNA comprising:

i.

 to effector strand that comprises

to.

 a first terminal effector nucleotide 5 ', wherein said first terminal effector nucleotide 5' comprises a first 2'-O-alkyl modification, and

b.

 a second terminal effector nucleotide 5 ', wherein said second terminal effector nucleotide 5' comprises a second 2'-O-alkyl modification; Y

ii.

 an antisense strand that comprises

to.

 a first 5 'terminal antisense nucleotide, wherein said first 5' terminal antisense nucleotide has one or more 5 'carbon-anchored phosphate groups of its sugar radical, and

b.

a second 5 'terminal antisense nucleotide, wherein said second 5' terminal antisense nucleotide comprises a third 2'-O-alkyl modification,

wherein said effector strand and said antisense strand are capable of forming a duplex of 18-19 base pairs of nucleotides having a 100% complementarity along the interval of the duplex, and within said duplex said first terminal effector nucleotide 5 'is the nucleotide plus 5' of the effector strand, said second terminal effector nucleotide 5 'is immediately adjacent and is downstream of the first terminal effector nucleotide 5', said first terminal antisense nucleotide 5 'is the nucleotide plus 5' of the strand antisense and said second terminal antisense nucleotide 5 'is immediately adjacent and is downstream of the first terminal antisense nucleotide 5', wherein all the nucleotides of the effector strand and the antisense strand other than said first terminal effector nucleotide 5 ', said second nucleotide 5 'terminal effector, and said second 5' terminal antisense nucleotide comprise a 2'-OH.

2.

 The double stranded siRNA of claim 1, wherein said first 2'-O-alkyl modification comprises 2'-Omethyl, said second 2'-O-alkyl modification comprises 2'-O-methyl, and said third modification 2 ' -O-alkyl comprises 2'-O-methyl.

3.

 The double stranded siRNA of claim 1, further comprising a 3 'overhang of 1 to 6 bases in at least one of said effector strand and said antisense strand.

Four.

 A double stranded siRNA as defined in any one of claims 1 to 3, for use in the treatment of the human or animal organism by therapy or for use in a diagnostic method that is implemented in the human organism or animal.