# Patent

## Title
Oligoribonucleotide inhibitors of NRF2 and methods of use thereof for treatment of cancer

## Abstract

The invention provides novel double stranded oligoribonucleotides that inhibit the Nrf2 gene. The invention also provides a pharmaceutical composition comprising one or more such oligoribonucleotides, and a vector capable of expressing the oligoribonucleotide. The present invention also relates to methods and compositions for treating or preventing the incidence or severity of a cancerous disease, particularly various lung cancers.

## Background
This application claims benefit of U.S. Provisional Application No. 60/937,135, filed Jun. 26, 2007 and of U.S. Provisional Application No. 60/919,431, filed Mar. 21, 2007, which contents of both are hereby incorporated by reference into this application.


Throughout this application various patent and scientific publications are cited. The disclosures for these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
This application contains nucleotide and/or amino acid sequences which are present on a compact disc labeled “OLIGORIBONUCLEOTIDE INHIBITORS OF NRF2 AND METHODS OF USE THEREOF FOR TREATMENT OF CANCER”. The file contained on this disc is named “Sequence Listing.txt”, is 347 KB in size, and was created on Mar. 19, 2008 in the IBM-PCT machine format and has operating system compatibility with MS-Windows. The entire content of this compact disc is hereby incorporated by reference into this application.
BACKGROUND OF THE INVENTION
siRNAs and RNA interference
RNA interference (RNAi) is a phenomenon involving double-stranded (ds) RNA-dependent gene specific posttranscriptional silencing. Originally, attempts to study this phenomenon and to manipulate mammalian cells experimentally were frustrated by an active, non-specific antiviral defence mechanism which was activated in response to long dsRNA molecules; see Gil et al. 2000, Apoptosis, 5:107-114. Later it was discovered that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without the stimulation of the generic antiviral defence mechanisms (see Elbashir et al. Nature 2001, 411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747). As a result, small interfering RNAs (siRNAs), which are short double-stranded RNAs, have become powerful tools in attempting to understand gene function.
Thus, RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) or microRNAs (miRNAs) (Ambros V. Nature 431:7006, 350-355 (2004); and Bartel D P. Cell. 2004 Jan. 23; 116 (2): 281-97 MicroRNAs: genomics, biogenesis, mechanism, and function). The corresponding process in plants is commonly referred to as specific post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. An siRNA is a double-stranded RNA molecule which down-regulates or silences (prevents) the expression of a gene/mRNA of its endogenous (cellular) counterpart. RNA interference is based on the ability of dsRNA species to enter a specific protein complex, where it is then targeted to the complementary cellular RNA and specifically degrades it. Thus, the RNA interference response features an endonuclease complex containing an siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA may take place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al 2001, Genes Dev., 15, 188). In more detail, longer dsRNAs are digested into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein et al., Nature, 2001, v. 409, p. 363-6; Lee et al., Nature, 2003, 425, p. 415-9). The RISC protein complex recognizes these fragments and complementary mRNA. The whole process is culminated by endonuclease cleavage of target mRNA (McManus&Sharp, Nature Rev Genet, 2002, v. 3, p. 737-47; Paddison &Hannon, Curr Opin Mol Ther. 2003 June; 5(3): 217-24). For information on these terms and proposed mechanisms, see Bernstein E., Denli A M. Hannon G J: 2001 The rest is silence. RNA. I; 7(11): 1509-21; Nishikura K.: 2001 A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. I 16; 107(4): 415-8 and PCT publication WO 01/36646 (Glover et al).
The selection and synthesis of siRNA corresponding to known genes has been widely reported; see for example Chalk A M, Wahlestedt C, Sonnhammer E L. 2004 Improved and automated prediction of effective siRNA Biochem. Biophys. Res. Commun. June 18; 319(1): 264-74; Sioud M, Leirdal M., 2004, Potential design rules and enzymatic synthesis of siRNAs, Methods Mol Biol.; 252:457-69; Levenkova N, Gu Q, Rux J J. 2004, Gene specific siRNA selector Bioinformatics. I 12; 20(3): 430-2. and Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K., Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference Nucleic Acids Res. 2004 I 9; 32(3):936-48.Se also Liu Y, Braasch D A, Nulf C J, Corey D R. Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 I 24; 43(7):1921-7. See also PCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al), and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemical modification analysis, RNA 2003 September; 9(9):1034-48 and I U.S. Pat. Nos. 5,898,031 and 6,107,094 (Crooke) for production of modified/more stable siRNAs.
Several groups have described the development of DNA-based vectors capable of generating siRNA within cells. The method generally involves transcription of short hairpin RNAs that are efficiently processed to form siRNAs within cells. Paddison et al. PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553. These reports describe methods to generate siRNAs capable of specifically targeting numerous endogenously and exogenously expressed genes.
Several studies have revealed that siRNA therapeutics are effective in vivo in both mammals and in humans. Bitko et al., have shown that specific siRNA molecules directed against the respiratory syncytial virus (RSV) nucleocapsid N gene are effective in treating mice when administered intranasally (Bitko et al., Nat. Med. 2005, 11(1):50-55). For reviews of therapeutic applications of siRNAs see for example, Barik (Mol. Med 2005, 83: 764-773); Chakraborty (Current Drug Targets 2007 8(3):469-82) and Dykxhoorn, et al (Gene Therapy 2006, 13, 541-552). Furthermore, a phase I clinical study with short siRNA molecule that targets the VEGFR1 receptor for the treatment of Age-Related Macular Degeneration (AMD) has been conducted in human patients. In studies such siRNA administered by intravitreal (intraocular) injection was found effective and safe in 14 patients tested (Kaiser, Am J Ophthalmol. 2006 142(4):660-8).
The Nrf2 Gene and Polypeptide (gi|166295208|ref|NM—006164.3| Homo sapiens Nuclear Factor (Erythroid-Derived 2)-Like 2 (NFE2L2):
Nuclear factor erythroid-2 related factor 2 (Nrf2), a cap-and-collar basic leucine zipper transcription factor, positively regulates a transcriptional program that maintains cellular redox homeostasis and protects cells from oxidative insult, including insult from chemotherapeutic agents (Rangasamy T, et al. J Clin Invest 114, 1248 (2004)). Nrf2 activates transcription of its target genes through binding specifically to the antioxidant-response element (ARE) found in those gene promoters. The Nrf2-regulated transcriptional program includes a broad spectrum of genes, including antioxidants such as heme oxygenase-1, superoxide dismutase, glutathione reductase (GSR), glutathione peroxidase, thioredoxin, thioredoxin reductase, peroxiredoxins (PRDX).
Lung Cancer:
Lung cancer is a cancer that forms in tissues of the lung, usually in the cells lining air passages. The two main types are small cell lung cancer and non-small cell lung cancer. These types are diagnosed based on the morphology of the cells under a microscope. It is the most lethal of all cancers worldwide, responsible for up to 3 million deaths annually. In non-small cell lung cancer (NSCLC), results of standard treatment are poor except for the most localized cancers. Surgery is the most potentially curative therapeutic option for this disease; radiation therapy can produce a cure in a small number of patients and can provide palliation in most patients. Adjuvant chemotherapy may provide an additional benefit to patients with resected NSCLC. In advanced-stage disease, chemotherapy offers modest improvements in median survival, though overall survival is poor. Chemotherapy has produced short-term improvement in disease-related symptoms.
WO 2006/128041 discloses specific siRNA molecules for Nrf2 and its use for treating any cancer, preferably lung and kidney cancers. US20020164576 discloses a method of inhibiting tumor growth (preferably a lymphoma cancer) using antisense molecules directed to Nrf2 or specific antibodies. US20070042418 discloses the use of siRNA molecules for Nrf2 for treating cancer.
Despite the evident progress, there remains a continued need for improved molecules in particular improved siRNA compounds able to treat cancerous diseases, particularly lung cancers.
SUMMARY OF THE INVENTION
The invention provides novel double stranded oligoribonucleotides that inhibit the Nrf2 gene. The invention also provides a pharmaceutical composition comprising one or more such oligoribonucleotides, and a vector capable of expressing the oligoribonucleotide. The present invention also relates to methods and compositions for treating or preventing the incidence or severity of a cancerous disease, particularly various lung cancers. The methods and compositions involve administering to a mammal in need of such treatment a prophylactically or therapeutically effective amount of one or more compounds which down-regulate expression of the Nrf2 gene, particularly novel small interfering RNAs (siRNAs), small molecule inhibitors of Nrf2 as described herein or antibodies to Nrf2 polypeptide.
In certain embodiments the present invention provides a compound having the structure:

5′(N)x—Z3′ (antisense strand)

3′Z′—(N′)y5′ (sense strand)

    
    
        wherein each of N and N′ is a ribonucleotide which may be independently modified or unmodified in its sugar residue;
        wherein each of (N)x and (N′)y is an oligomer in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond;
        wherein each of x and y is an integer between 19 and 40;
        wherein each of Z and Z′ may be present or absent, but if present is 1-5 consecutive nucleotides covalently attached at the 3′ terminus of the strand in which it is present; and
        wherein the sequence of (N)x is set forth as any one of SEQ ID NOS 504 to 1006 or 1507 to 2006, and the sequence of (N′)y is set forth as the complementary sequence. The sequence of (N′)y is a complementary sequence set forth as SEQ ID NOS: 1 to 503 or 1007 to 1506, respectively.
    
    


In some embodiments the covalent bond joining each consecutive N or N′ is a phosphodiester bond. In various embodiments all the covalent bonds are phosphodiester bonds.
In various embodiments the compound comprises ribonucleotides wherein x=y and each of x and y is 19, 20, 21, 22 or 23. In some embodiments x=y=23. In other embodiments x=y=19.
In some embodiments the compound is blunt ended, for example wherein both Z and Z′ are absent. In an alternative embodiment, the compound comprises at least one 3′ overhang, wherein at least one of Z or Z′ is present. Z and Z′ can independently comprise one or more covalently linked modified or non-modified nucleotides, for example inverted dT or dA; dT, LNA, mirror nucleotide and the like. In some embodiments each of Z and Z′ are independently selected from dT and dTdT.
In some embodiments N or N′ comprises a modification in the sugar residue of one or more ribonucleotides. In other embodiments the compound comprises at least one ribonucleotide modified in the sugar residue. In some embodiments the compound comprises a modification at the 2′ position of the sugar residue. In some embodiments the modification in the 2′ position comprises the presence of an amino, a fluoro, an alkoxy or an alkyl moiety. In certain embodiments the 2′ modification comprises a methoxy moiety. A presently preferred modification is a 2′ methoxy of the sugar residue (2′-O-methyl; 2′-O-Me; 2′-O—CH3).
In some embodiments the compound comprises modified alternating ribonucleotides in one or both of the antisense and the sense strands. In certain embodiments the compound comprises modified alternating ribonucleotides in the antisense and the sense strands. In other embodiments the compound comprises modified alternating ribonucleotides in the antisense strand only. In certain embodiments the middle ribonucleotide of the antisense strand is not modified; e.g. ribonucleotide in position 10 in a 19-mer strand or position 12 in a 23-mer strand.
In additional embodiments the compound comprises modified ribonucleotides in alternating positions wherein each N at the 5′ and 3′ termini of (N)x are modified in their sugar residues, and each N′ at the 5′ and 3′ termini of (N′)y are unmodified in their sugar residues. In some embodiments, neither (N)x nor (N′)y are phosphorylated at the 3′ and 5′ termini. In other embodiments either or both (N)x and (N′)y are phosphorylated at the 3′ termini. Preferably the siRNA molecule is either phosphorylated at the 3′ termini of both sense and anti-sense strands, or non-phosphorylated at all; similar results are obtained for both siRNAs. The siRNA molecules used in the in vitro experiments were phosphorylated at the 3′ termini of both sense and anti-sense strands while the siRNA molecules used in the in vivo experiments were non-phosphorylated at all.
More specifically, the present invention provides methods and compositions for treating a patient suffering from a cancerous disease, (e.g. lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer). In a particular embodiment, the cancer is lung cancer such as non-small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma. The methods of the invention comprising administering to the patient one or more compounds which down-regulate expression of the Nrf2 gene, particularly siRNAs that inhibit Nrf2, typically as a pharmaceutical composition, in a therapeutically effective dose so as to thereby treat the patient.
Still further, the invention relates to a method for treating or preventing the incidence or severity of a cancerous disease, particularly a lung disease in a patient comprising administering to the patient a composition comprising an effective amount of naked siRNA molecules. Preferably, the naked siRNA molecules are applied directly to lung for example via aerosol delivery which has the potential for delivering high concentrations of the therapeutic molecule to the inner lung.
Still further, the invention relates to the use of a therapeutically effective dose of an oligonucleotide for the preparation of a composition for treating a subject suffering from a cancerous disease, preferably lung cancer, wherein the oligonucleotide is set forth as any one of SEQ ID NOS: 1-2006.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: A549-C8 luciferase cells were transfected with non-targeting luciferase siRNA or Nrf2 siRNA-1 (sense strand SEQ ID NO: 10) at 20 nM concentration. Seventy two hours post transfection, cells were harvested and total RNA was isolated. Nrf2 knockdown as well as expression of two Nrf2 dependent target genes (GCLM and HO-1) were analyzed by real time RT-PCR.
FIG. 2: SCID-Beige mice injected i.v. with ARE-luciferase reporter tumor cells were inhaled with Nrf2 siRNA (sense strand SEQ ID NO: 10) twice during the 4th week of lung tumor growth. Control mice were inhaled with GFP siRNA. Mice were imaged before and after siRNA inhalation.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates generally to compounds which down-regulate expression of the Nrf2 gene particularly to novel small interfering RNAs (siRNAs), and to the use of these novel siRNAs in the treatment of various diseases and medical conditions in particular various forms of cancerous diseases. Such preferred siRNA compounds comprise sequences set forth as any one of SEQ ID NOS: 1 to 2006.
The inventors of the present invention have provided novel siRNAs to Nrf2 in order to treat any of the above diseases or disorders. Methods, molecules and compositions which inhibit Nrf2 are discussed herein at length, and any of said molecules and/or compositions may be beneficially employed in the treatment of a patient suffering from any of said conditions.
The present invention provides methods and compositions for inhibiting expression of a target Nrf2 gene in vivo. In general, the method includes administering oligoribonucleotides, such as small interfering RNAs (i.e., siRNAs) that are targeted to a particular Nrf2 mRNA and hybridize to, or interact with, the mRNAs under biological conditions (within the cell), or a nucleic acid material that can produce siRNA in a cell, in an amount sufficient to down-regulate expression of a target gene by an RNA interference mechanism. In particular, the subject method can be used to inhibit expression of the Nrf2 gene for treatment of a disease.
In accordance with the present invention, the siRNA molecules or inhibitors of the Nrf2 gene may be used as drugs to treat various pathologies accompanied by an elevated level of Nrf2 polypeptide.
The present invention provides double-stranded oligoribonucleotides (siRNAs), which down-regulate the expression of the Nrf2 gene. An siRNA of the invention is a duplex oligoribonucleotide in which the sense strand is derived from the mRNA sequence of the Nrf2 gene, and the antisense strand is complementary to the sense strand. In general, some deviation from the target mRNA sequence is tolerated without compromising the siRNA activity (see e.g. Czauderna et al 2003 Nucleic Acids Research 31(11), 2705-2716). An siRNA of the invention inhibits gene expression on a post-transcriptional level with or without destroying the mRNA. Without being bound by theory, siRNA may target the mRNA for specific cleavage and degradation and/or may inhibit translation from the targeted message.
As used herein, the term “Nrf2”, or “Nrf2 polypeptide” (gi|166295208|ref|NM—006164.3| Homo sapiens nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) is defined as any homolog of the Nrf2 polypeptide having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to the Nrf2 gene, as either full-length or a fragment or a domain thereof, as a mutant or the polypeptide encoded by a spliced variant nucleic acid sequence, as a chimera with other polypeptides, provided that any of the above has the same or substantially the same biological function as the Nrf2 polypeptide.
Generally, the siRNAs used in the present invention comprise a ribonucleic acid comprising a double stranded structure, whereby the double-stranded structure comprises a first strand and a second strand, whereby the first strand comprises a first stretch of contiguous nucleotides and whereby said first stretch is at least partially complementary to a target nucleic acid, and the second strand comprises a second stretch of contiguous nucleotides and whereby said second stretch is at least partially identical to a target nucleic acid, whereby said first strand and/or said second strand comprises a plurality of groups of modified nucleotides having a modification at the 2′-position whereby within the strand each group of modified nucleotides is flanked on one or both sides by a flanking group of nucleotides whereby the flanking nucleotides forming the flanking group of nucleotides is either an unmodified nucleotide or a nucleotide having a modification different from the modification of the modified nucleotides. Further, said first strand and/or said second strand may comprise said plurality of modified nucleotides and may comprises said plurality of groups of modified nucleotides.
The group of modified nucleotides and/or the group of flanking nucleotides may comprise a number of nucleotides whereby the number is selected from the group comprising one nucleotide to 10 nucleotides. In connection with any ranges specified herein it is to be understood that each range discloses any individual integer between the respective figures used to define the range including said two figures defining said range. In the present case the group thus comprises one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides and ten nucleotides.
The pattern of modified nucleotides of said first strand may be shifted by one or more nucleotides relative to the pattern of modified nucleotides of the second strand.
The modifications discussed above may be selected from the group comprising amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, thiolate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2, N3; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described in European patents EP 0 586 520 B1 or EP 0 618 925 B1.
The double stranded structure of the siRNA may be blunt ended, on one or both sides. More specifically, the double stranded structure may be blunt ended on the double stranded structure's side which is defined by the 5′-end of the first strand and the 3′-end of the second strand, or the double stranded structure may be blunt ended on the double stranded structure's side which is defined by at the 3′-end of the first strand and the 5′-end of the second strand.
Additionally, at least one of the two strands may have an overhang of at least one nucleotide at the 5′-end; the overhang may consist of at least one deoxyribonucleotide. At least one of the strands may also optionally have an overhang of at least one nucleotide at the 3′-end.
The length of the double-stranded structure of the siRNA is typically from about 17 to 21 and more preferably 18 or 19 bases. Further, the length of said first strand and/or the length of said second strand may independently from each other be selected from the group comprising the ranges of from about 15 to about 23 bases, 17 to 21 bases and 18 or 19 bases.
Additionally, the complementarily between said first strand and the target nucleic acid may be perfect, or the duplex formed between the first strand and the target nucleic acid may comprise at least 15 nucleotides wherein there is one mismatch or two mismatches between said first strand and the target nucleic acid forming said double-stranded structure.
Substantially complementary refers to complementarity of greater than about 84%, to another sequence. For example in a duplex region consisting of 19 base pairs one mismatch results in 94.7% complementarity, two mismatches results in about 89.5% complementarity and 3 mismatches results in about 84.2% complementarity, rendering the duplex region substantially complementary. Accordingly substantially identical refers to identity of greater than about 84%, compared to another sequence.
In some cases both the first strand and the second strand each comprise at least one group of modified nucleotides and at least one flanking group of nucleotides, whereby each group of modified nucleotides comprises at least one nucleotide and whereby each flanking group of nucleotides comprising at least one nucleotide with each group of modified nucleotides of the first strand being aligned with a flanking group of nucleotides on the second strand, whereby the most terminal 5′ nucleotide of the first strand is a nucleotide of the group of modified nucleotides, and the most terminal 3′ nucleotide of the second strand is a nucleotide of the flanking group of nucleotides. Each group of modified nucleotides may consist of a single nucleotide and/or each flanking group of nucleotides may consist of a single nucleotide.
Additionally, it is possible that on the first strand the nucleotide forming the flanking group of nucleotides is an unmodified nucleotide which is arranged in a 3′ direction relative to the nucleotide forming the group of modified nucleotides, and on the second strand the nucleotide forming the group of modified nucleotides is a modified nucleotide which is arranged in 5′ direction relative to the nucleotide forming the flanking group of nucleotides.
Further the first strand of the siRNA may comprise eight to twelve, preferably nine to eleven, groups of modified nucleotides, and the second strand may comprise seven to eleven, preferably eight to ten, groups of modified nucleotides.
The first strand and the second strand may be linked by a loop structure, which may be comprised of a non-nucleic acid polymer such as, inter alia, polyethylene glycol. Alternatively, the loop structure may be comprised of a nucleic acid.
Further, the 5′-terminus of the first strand of the siRNA may be linked to the 3′-terminus of the second strand, or the 3′-end of the first strand may be linked to the 5′-terminus of the second strand, said linkage being via a nucleic acid linker typically having a length between 10-2000 nucleobases.
In particular, the invention provides a compound having structure A:










5′ (N)x-Z 3′
(antisense strand)



 



3′ Z′-(N′)y 5′
(sense strand)






    
    
        wherein each N and N′ is a ribonucleotide which may independently be modified or unmodified in its sugar residue and each of (N)x and (N′)y is oligomer in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond;
        wherein each of x and y is an integer between 19 and 40;
        wherein each of Z and Z′ may be present or absent, but if present is dTdT and is covalently attached at the 3′ terminus of the strand in which it is present;
        and wherein the sequence of (N)x is set forth as any one of SEQ ID NOS 504 to 1006 or 1507 to 2006, and the sequence of (N′)y is set forth as the complementary sequence. The sequence of (N′)y is a complementary sequence set forth as SEQ ID NOS: 1 to 503 or 1007 to 1506, respectively.
    
    


It will be readily understood by those skilled in the art that the compounds of the present invention consist of a plurality of nucleotides, which are linked through covalent linkages. Each such covalent linkage may be a phosphodiester linkage, a phosphothioate linkage, or a combination of both, along the length of the nucleotide sequence of the individual strand.
Other possible backbone modifications are described inter alia in U.S. Pat. Nos. 5,587,361; 6,242,589; 6,277,967; 6,326,358; 5,399,676; 5,489,677; and 5,596,086.
In particular embodiments, x and y are preferably an integer between about 19 to about 27, most preferably from about 19 to about 23. In a particular embodiment of the compound of the invention, x may be equal to y (viz., x=y) and in preferred embodiments x=y=19 or x=y=21. In a particularly preferred embodiment x=y=19.
In one embodiment of the compound of the invention, Z and Z′ are both absent; in another embodiment one of Z or Z′ is present.
In one embodiment of the compound of the invention, all of the ribonucleotides of the compound are unmodified in their sugar residues.
In preferred embodiments of the compound of the invention, at least one ribonucleotide is modified in its sugar residue, preferably a modification at the 2′ position. The modification at the 2′ position results in the presence of a moiety which is preferably selected from the group comprising amino, fluoro, methoxy, alkoxy and alkyl groups. In a presently most preferred embodiment the moiety at the 2′ position is methoxy (2′-O-methyl).
In preferred embodiments of the invention, alternating ribonucleotides are modified in both the antisense and the sense strands of the compound. In particular the siRNA used in the Examples has been such modified such that a 2′ O-Me group was present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i. e. a 2′-O-Me group was present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand. Additionally, it is to be noted that the in case of these particular nucleic acids according to the present invention the first stretch is identical to the first strand and the second stretch is identical to the second strand and these nucleic acids are also blunt ended.
According to one preferred embodiment of the invention, the antisense and the sense strands of the siRNA molecule are both phosphorylated only at the 3′-terminus and not at the 5′-terminus. According to another preferred embodiment of the invention, the antisense and the sense strands are both non-phosphorylated both at the 3′-terminus and also at the 5′-terminus. According to yet another preferred embodiment of the invention, the 1st nucleotide in the 5′ position in the sense strand is specifically modified to abolish any possibility of in vivo 5′-phosphorylation.
In another embodiment of the compound of the invention, the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues.
The invention further provides a vector capable of expressing any of the aforementioned oligoribonucleotides in unmodified form in a cell after which appropriate modification may be made.
The invention also provides a composition comprising one or more of the compounds of the invention in a carrier, preferably a pharmaceutically acceptable carrier. This composition may comprise a mixture of two or more different siRNAs.
The invention also provides a composition which comprises the above compound of the invention covalently or non-covalently bound to one or more compounds of the invention in an amount effective to inhibit Nrf2 and a carrier. This composition may be processed intracellularly by endogenous cellular complexes to produce one or more oligoribonucleotides of the invention.
The invention also provides a composition comprising a carrier and one or more of the compounds of the invention in an amount effective to down-regulate expression in a cell of Nrf2, which compound comprises a sequence substantially complementary to the sequence of (N)x.
Additionally the invention provides a method of down-regulating the expression of Nrf2 by at least 50% as compared to a control comprising contacting an mRNA transcript of Nrf2 with one or more of the compounds of the invention.
In one embodiment the oligoribonucleotide is down-regulating Nrf2, whereby the down-regulation of Nrf2 is selected from the group comprising down-regulation of gene function, down-regulation of polypeptide and down-regulation of mRNA expression.
In one embodiment the compound is down-regulating the Nrf2 polypeptide, whereby the down-regulation is selected from the group comprising down-regulation of function (which may be examined by an enzymatic assay or a binding assay with a known interactor of the native gene/polypeptide, inter alia), down-regulation of protein (which may be examined by Western blotting, ELISA or immuno-precipitation, inter alia) and down-regulation of mRNA expression (which may be examined by Northern blotting, quantitative RT-PCR, in-situ hybridisation or microarray hybridisation, inter alia).
The invention also provides a method of treating a patient suffering from a disease accompanied by an elevated level of Nrf2, the method comprising administering to the patient a composition of the invention in a therapeutically effective dose thereby treating the patient.
More particularly, the invention provides an oligoribonucleotide wherein one strand comprises consecutive nucleotides having, from 5′ to 3′, the sequence set forth in Table A (19 mer siRNA molecules, SEQ ID NOS: 1-1006) or in Table B (21 mer siRNA molecules, SEQ ID NOS: 1007-2006), or a homolog thereof wherein in up to 2 of the nucleotides in each terminal region a base is altered. Preferred 19 mer siRNA molecules comprise the sense and corresponding antisense sequences listed in Table C. More preferred siRNA comprises SEQ ID NO: 10 (sense sequence) and SEQ ID NO: 513 (antisense sequence).
The terminal region of the oligonucleotide refers to bases 1-4 and/or 16-19 in the 19-mer sequence and to bases 1-4 and/or 18-21 in the 21-mer sequence.
The presently most preferred compound of the invention is a blunt-ended 19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent. The oligonucleotide molecule is either phosphorylated at 3′ termini of both sense and anti-sense strands, or non-phosphorylated at all; or having 1st nucleotide in the 5′ position on the sense strand specifically modified to abolish any possibility of in vivo 5′-phosphorylation. The alternating ribonucleotides are modified at the 2′ position in both the antisense and the sense strands, wherein the moiety at the 2′ position is methoxy (2′-O-methyl) and wherein the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues.
In certain embodiments the present invention provides a compound having the structure










5′ (N)x 3′
antisense strand



 



3′ (N′)y 5′
sense strand






    
    
        wherein each of x and y=19 and (N)x and (N′)y are fully complementary;
        wherein alternating ribonucleotides in (N)x and (N′)y are modified to result in a 2′-O-methyl modification in the sugar residue of the ribonucleotides;
        wherein each N at the 5′ and 3′ termini of (N)x are modified;
        wherein each N′ at the 5′ and 3′ termini of (N′)y are unmodified;
        wherein each of (N)x and (N′)y is selected from the group of oligomers set forth in any one of Tables A and B (SEQ ID NOS: 1-2006).
        (N)x and (N′)y may be phosphorylated or non-phosphorylated at the 3′ and 5′ termini.
    
    


In certain embodiments of the invention, alternating ribonucleotides are modified in the 2′ position of the sugar residue in both the antisense and the sense strands of the compound. In particular the siRNAs may be modified such that a 2′-O-methyl (Me) group is present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i. e. a 2′-O-Me group, is present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand. These particular siRNA compounds may also be blunt ended.
In certain embodiments of the compounds of the invention having alternating ribonucleotides modified in one or both of the antisense and the sense strands of the compound; for 19-mers and 23-mers the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues. For 21-mers the ribonucleotides at the 5′ and 3′ termini of the sense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the antisense strand are unmodified in their sugar residues. As mentioned above, it is preferred that the middle nucleotide of the antisense strand is unmodified.
In some preferred embodiments the middle ribonucleotide in the first strand (antisense) is an unmodified nucleotide. For example, in a 19-oligomer antisense strand, ribonucleotide number 10 is unmodified; in a 21-oligomer antisense strand, ribonucleotide number 11 is unmodified; and in a 23-oligomer antisense strand, ribonucleotide number 12 is unmodified. The modifications or pattern of modification, if any, of the siRNA must be planned to allow for this.
Delivery: The siRNA molecules of the present invention may be delivered to the target tissue (such as the lung) by direct application of the naked molecules admixed with a carrier or a diluent using an aerosol.
For administration via the upper respiratory tract, the composition is formulated into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a suspension, at an appropriate concentration for oro-nasal administration as an aerosol. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's Pharmaceutical Sciences 16th edition, Ed. Arthur Osol, page 1445 (1980)). One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and/or upper respiratory administration.
Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, “minor amounts” means no auxiliary agents or substances are present that might affect or mediate uptake of nucleic acid in the cells of the lungs.
Aerosol dosage, formulations and delivery systems may be selected for a particular therapeutic application, as described, for example, in Gonda, I. “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313, 1990; and in Moren, “Aerosol dosage forms and formulations,” in: Aerosols in Medicine, Principles, Diagnosis and Therapy, Moren, et al., Eds. Esevier, Amsterdam, 1985. The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques, such as ultrasonication or high pressure treatment.
Various suitable devices and methods of inhalation which can be used to administer particles to a patient's respiratory tract are known in the art. Nebulizers create a fine mist from a solution or suspension, which is inhaled by the patient. The devices described in U.S. Pat. No. 5,709,202 to Lloyd, et al., can be used. An MDI typically includes a pressurized canister having a meter valve, wherein the canister is filled with the solution or suspension and a propellant. The solvent itself may function as the propellant, or the composition may be combined with a propellant, such as freon. The composition is a fine mist when released from the canister due to the release in pressure. The propellant and solvent may wholly or partially evaporate due to the decrease in pressure.
The term “naked siRNA” refers to siRNA molecules that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. For example, siRNA in PBS is “naked siRNA”. However, the siRNA molecules of the invention can also be delivered in liposome formulations and lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed, see, for example, Shen et al (FEBS letters 539: 111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002), Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J. Mol. Biol. 327: 761-766 (2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni et al., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recently been successfully used for inhibition in primates; for further details see Tolentino et al., Retina 24(1) February 2004 I 132-138. Respiratory formulations for siRNA are described in U.S. patent application No. 2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and other steroid and lipid conjugated siRNAs) can been used for delivery see Soutschek et al Nature 432: 173-177(2004); and Lorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004).
The siRNAs or pharmaceutical compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the disease to be treated, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
The “therapeutically effective dose” for purposes herein is thus determined by such considerations as are known in the art. The dose must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. The compounds of the present invention can be administered by any of the conventional routes of administration. It should be noted that the compound can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles. The compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful. Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, and other parental routes of administration. The liquid compositions include aqueous solutions, with and without organic co-solvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles. In addition, under certain circumstances the compositions for use in the novel treatments of the present invention may be formed as aerosols, for intranasal and like administration. The patient being treated is a warm-blooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention and they include liposomes and microspheres.
Examples of delivery systems useful in the present invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art. In one specific embodiment of this invention topical and transdermal formulations are particularly preferred.
In general, the active dose of compound for humans is in the range of from 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about 0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of one dose per day or twice or three or more times per day for a period of 1-4 weeks or longer.
The term “treatment” as used herein refers to administration of a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring. Thus “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a disease in particular a cancerous disease such as lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer. In a particular embodiment, the cancer is lung cancer such as non-small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma
In another aspect of the invention a pharmaceutical composition is provided which comprises any of the above oligoribonucleotides or vectors and a pharmaceutically acceptable carrier. Another aspect of the invention is the use of a therapeutically effective amount of any of the above oligoribonucleotides or vectors for the preparation of a medicament for treating a patient suffering from a cancer disease.
By “cancerous disease” is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Examples of cancerous diseases include, without limitation, leukemias (e. g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio sarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyo sarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, crailiopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwamioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).
In one embodiment, the present invention provides a pharmaceutical composition comprising an Nrf2 inhibitory molecule, preferable an siRNA molecule that decreases the expression of the Nrf2 gene in combination with a chemotherapeutic agent. Without being bound by theory, since Nrf2 positively regulates drug detoxification enzymes, targeting this molecule may have a broad effect on all anticancer drugs. In various embodiments, the Nrf2 inhibitory molecule is administered prior to, concurrently with, or following administration of a chemotherapeutic. Without wishing to be bound by theory, administration of an Nrf2 inhibitory molecule likely enhances the accumulation or efficacy of a chemotherapeutic agent.
Compositions and methods of the invention may be used in combination with any conventional therapy known in the art. In one embodiment, the Nrf2 inhibitory molecules of the invention may be used in combination with one or more anti-cancerous therapy known in the art. Thus the Nrf2 inhibitors may be used before, during or following anti-cancerous therapy Exemplary anti-cancerous therapies include, for example, chemotherapy, cryotherapy, hormone therapy, growth factor inhibitors, radiotherapy, and surgery. Chemotherapeutics typically used in the treatment of a cancerous disease are for example abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide, cachectin, cernadotin, chorambucil, cyclophosphamide, 3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNTJ), cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, oxaliplatin paclitaxel, prednimustine, procarbazine, RPR1 09881, satrapaltin, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine. Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Heliman (editors), 6th edition (Fel. 15, 2001), Lippincott Williams & Wilkins Publishers. A preferred combination according to the present invention is an siRNA molecule targeting Nrf2 with platinum drugs, more preferably an siRNA molecule set forth in Table C with a platinum drug, preferably carboplatin. Without being bound by theory, the Nrf2 siRNA inhibitors of the invention are found to be efficient promoters for the antineoplastic potential of platinum drugs, causing additive/synergistic effects in cancer cells. Platinum drugs include carboplatin, cisplatin, oxaliplatin and satrapaltin inter alia; see Kelland and Farrell, Platinum-based drugs in cancer therapy (Cancer drug discovery & development) Lavoisier 2000 which is hereby incorporated by reference.
The compounds which reduce or prevent the cancerous disease such as lung cancer, eg the novel siRNAs inter alia are preferably administered directly to the inner lung as naked siRNA in a vehicle such as PBS or other physiological solutions, but may alternatively be administered with a delivery vehicle as described above.
The present invention also provides for a process of preparing a pharmaceutical composition, which comprises:

    
    
        obtaining one or more double stranded compound of the invention; and
        admixing said compound with a pharmaceutically acceptable carrier.
    
    


The present invention also provides for a process of preparing a pharmaceutical composition, which comprises admixing one or more compounds of the present invention with a pharmaceutically acceptable carrier.
In a preferred embodiment, the compound used in the preparation of a pharmaceutical composition is admixed with a carrier in a pharmaceutically effective dose. In a particular embodiment the compound of the present invention is conjugated to a steroid or to a lipid or to another suitable molecule e.g. to cholesterol.
Modifications or analogs of nucleotides can be introduced to improve the therapeutic properties of the nucleotides. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes.
Accordingly, the present invention also includes all analogs of, or modifications to, a oligonucleotide of the invention that does not substantially affect the function of the polynucleotide or oligonucleotide. In a preferred embodiment such modification is related to the base moiety of the nucleotide, to the sugar moiety of the nucleotide and/or to the phosphate moiety of the nucleotide.
In embodiments of the invention, the nucleotides can be selected from naturally occurring or synthetically modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Modified bases of the oligonucleotides include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl-adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thioalkyl adenine, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
In addition, analogs of nucleotides can be prepared wherein the structures of the nucleotides are fundamentally altered and are better suited as therapeutic or experimental reagents. An example of a nucleotide analog is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone similar to that found in peptides. PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. Further, PNAs have been shown to bind more strongly to a complementary DNA sequence than to a DNA molecule. This observation is attributed to the lack of charge repulsion between the PNA strand and the DNA strand. Other modifications that can be made to oligonucleotides include polymer backbones, cyclic backbones, or acyclic backbones.
In one embodiment the modification is a modification of the phosphate moiety, whereby the modified phosphate moiety is selected from the group comprising phosphothioate.
The compounds of the present invention can be synthesized by any of the methods that are well-known in the art for synthesis of ribonucleic (or deoxyribonucleic) oligonucleotides. Such synthesis is, among others, described in Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48: 2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49: 6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154: 287-313; the synthesis of thiolates is, among others, described in Eckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules is described in Sproat B., in Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 and respective downstream processes are, among others, described in Pingoud A. et. al., in IRL Press 1989 edited by Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 (supra).
Other synthetic procedures are known in the art e.g. the procedures as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684; and Wincott et al., 1997, Methods Mol. Bio., 74, 59, and these procedures may make use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The modified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotides are incorporated as desired.
The oligonucleotides of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
It is noted that a commercially available machine (available, inter alia, from Applied Biosystems) can be used; the oligonucleotides are prepared according to the sequences disclosed herein. Overlapping pairs of chemically synthesized fragments can be ligated using methods well known in the art (e.g., see U.S. Pat. No. 6,121,426). The strands are synthesized separately and then are annealed to each other in the tube. Then, the double-stranded siRNAs are separated from the single-stranded oligonucleotides that were not annealed (e.g. because of the excess of one of them) by HPLC. In relation to the siRNAs or siRNA fragments of the present invention, two or more such sequences can be synthesized and linked together for use in the present invention.
The compounds of the invention can also be synthesized via a tandem synthesis methodology, as described in US patent application publication No. US2004/0019001 (McSwiggen), wherein both siRNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siRNA fragments or strands that hybridize and permit purification of the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker.
The present invention further provides for a pharmaceutical composition comprising two or more siRNA molecules for the treatment of any of the diseases and conditions mentioned herein, whereby said two molecules may be physically mixed together in the pharmaceutical composition in amounts which generate equal or otherwise beneficial activity, or may be covalently or non-covalently bound, or joined together by a nucleic acid linker of a length ranging from 2-100, preferably 2-50 or 2-30 nucleotides. In one embodiment, the siRNA molecules are comprised of a double-stranded nucleic acid structure as described herein, wherein the two siRNA sequences are selected from Tables A or B.
In another embodiment, the siRNA molecules are comprised of a double-stranded nucleic acid structure, wherein the first siRNA sequence is selected from Tables A or B, and the second siRNA molecule targets another anti-cancer gene, thereby providing beneficial activity. The tandem double-stranded structure which comprises two or more siRNA sequences is processed intracellularly to form two or more different siRNAs. Such second siRNA molecule is preferably an siRNA molecule that targets a anti-cancer gene. Preferred anti-cancer genes are for example gene encoding growth factors such as insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet derived growth factor (PDGF).
The siRNA molecules are covalently or non-covalently bound or joined by a linker to form a tandem siRNA molecule. Such tandem siRNA molecules comprising two siRNA sequences are typically of 38-150 nucleotides in length, more preferably 38 or 40-60 nucleotides in length, and longer accordingly if more than two siRNA sequences are included in the tandem molecule. A longer tandem molecule comprised of two or more longer sequences which encode siRNA produced via internal cellular processing, e.g., long dsRNAs, is also envisaged, as is a tandem molecule encoding two or more shRNAs. Such tandem molecules are also considered to be a part of the present invention.
siRNA molecules that target Nrf2 may be the main active component in a pharmaceutical composition, or may be one active component of a pharmaceutical composition containing two or more siRNAs (or molecules which encode or endogenously produce two or more siRNAs, be it a mixture of molecules or one or more tandem molecules which encode two or more siRNAs), said pharmaceutical composition further being comprised of one or more additional siRNA molecule which targets one or more additional gene. Simultaneous inhibition of Nrf2 and said additional gene(s) will likely have an additive or synergistic effect for treatment of the diseases disclosed herein.
In a preferred embodiment, the one or more additional siRNA molecules target an anti-cancer gene, thus having an additive or synergistic effect with the Nrf2 siRNA. The additional siRNA molecules may target one or more of the anti-cancer genes defined above.
As disclosed herein, aptamers may also be used in the present invention alone or in combination with the novel siRNAs disclosed herein for targeting NRF2 and for the treatment of any one of the conditions disclosed herein. For example, an aptamer can be used with any one of the siRNAs disclosed herein in combination therapy for the treatment of any one of the conditions disclosed herein. The novel pharmaceutical composition employed for such a combination therapy, which is also part of the present invention, may comprise an siRNA of the present invention covalently or non-covalently attached to an aptamer. Aptamers are RNA or DNA single-strand or double-strand oligonucleic acids which bind to a target protein and do not generally exhibit non-specific effects. Aptamers can be modified for stability or other desired qualities in accordance with any nucleic acid modifications disclosed herein and/or known to one of skill in the art. Modifications to aptamers can be introduced anywhere in the molecule, such as the 5′ or 3′ termini, or at any internally defined modification site. For example, RNA aptamers can be stabilized with 2′-fluoro or 2′-amino modified pyrimidines. Aptamers can also be linked to reporter molecules or linker chemistries and can be attached to beads or other solid support if necessary (e.g., 5′ or 3′ amino, thiol ester or biotin groups). Thioaptamers are aptamers which contain sulfur modifications at specific internucleoside phosphoryl sites, and may possess enhanced stability, nuclease resistance, target affinity and/or selectivity. Examples of thioaptamers include phosphoromonothioate (S-ODN) and phosphorodithioate (S2-ODN) oligodeoxy thioaptamers. For further information on aptamers and thioaptamers see U.S. Pat. Nos. 5,218,088 and 6,423,493.
Additionally, the siRNA disclosed herein or any nucleic acid molecule comprising or encoding such siRNA can be linked or bound (covalently or non-covalently) to antibodies (including aptamer molecules) against cell surface internalizable molecules expressed on the target cells, in order to achieve enhanced targeting for treatment of the diseases disclosed herein. For example, anti-Fas antibody (preferably a neutralizing antibody) may be combined (covalently or non-covalently) with a Nrf2 siRNA molecule. In another example, an aptamer which can act like a ligand/antibody may be combined (covalently or non-covalently) with a Nrf2 siRNA molecule.
The term “Covalent bonding” as used herein refers to chemical bonding that is characterized by the sharing of pairs of electrons between atoms.
The term “Noncovalent bonding” as used herein refers to a variety of interactions that are not covalent in nature between molecules or parts of molecules that provide force to hold the molecules or parts of molecules together, usually in a specific orientation or conformation. These noncovalent interactions include: ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces and dipole-dipole bonds.
The compounds of the present invention can be delivered either directly or with viral or non-viral vectors. When delivered directly the sequences are generally rendered nuclease resistant. Alternatively the sequences can be incorporated into expression cassettes or constructs such that the sequence is expressed in the cell as discussed herein below. Generally the construct contains the proper regulatory sequence or promoter to allow the sequence to be expressed in the targeted cell. Vectors optionally used for delivery of the compounds of the present invention are commercially available, and may be modified for the purpose of delivery of the compounds of the present invention by methods known to one of skill in the art.
It is also envisaged that a long oligonucleotide (typically 25-500 nucleotides in length) comprising one or more stem and loop structures, where stem regions comprise the sequences of the oligonucleotides of the invention, may be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and may be processed intracellularly by endogenous cellular complexes (e.g. by DROSHA and DICER as described above) to produce one or more smaller double stranded oligonucleotides (siRNAs) which are oligonucleotides of the invention. This oligonucleotide can be termed a tandem shRNA construct. It is envisaged that this long oligonucleotide is a single stranded oligonucleotide comprising one or more stem and loop structures, wherein each stem region comprises a sense and corresponding antisense siRNA sequence of an NRF2 gene. In particular, it is envisaged that this oligonucleotide comprises sense and antisense siRNA sequences as depicted as any one of SEQ ID NO: 1-2006.
As used herein, the term “inhibition” of the Nrf2 gene means inhibition of the gene expression (transcription or translation) or polypeptide activity.
Although the inhibitor may be an siRNA molecule, other inhibitors contemplated to be used in the methods of the invention to inhibit the Nrf2 gene and to treat the diseases and conditions described herein are inter alia antibodies, preferably neutralizing antibodies or fragments thereof, including single chain antibodies, antisense oligonucleotides, antisense DNA or RNA molecules, aptamers, proteins, polypeptides and peptides including peptido-mimetics and dominant negatives, and also expression vectors expressing all the above. Additional inhibitors may be small chemical molecules, which generally have a molecular weight of less than 2000 daltons, more preferably less than 1000 daltons, even more preferably less than 500 daltons. These inhibitors may act as follows: small molecules may affect expression and/or activity; antibodies may affect activity; all kinds of antisense may affect the gene expression; and dominant negative polypeptides and peptidomimetics may affect activity; expression vectors may be used inter alia for delivery of antisense or dominant-negative polypeptides or antibodies.
The term “antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody, inter alia. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of antibodies comprising an antigen-binding domain, e.g. antibodies without the Fc portion, single chain antibodies, miniantibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc. The term “antibody” may also refer to antibodies against polynucleotide sequences obtained by cDNA vaccination. The term also encompasses antibody fragments which retain the ability to selectively bind with their antigen or receptor and are exemplified as follows, inter alia:

    
    
        (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule which can be produced by digestion of whole antibody with the enzyme papain to yield a light chain and a portion of the heavy chain;
        (2) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′2) is a dimer of two Fab fragments held together by two disulfide bonds;
        (3) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
        (4) Single chain antibody (SCA), defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule.
    
    


Antisense Molecules
By the term “antisense” (AS) or “antisense fragment” is meant a polynucleotide fragment (comprising either deoxyribonucleotides, ribonucleotides or a mixture of both) having inhibitory antisense activity, said activity causing a decrease in the expression of the endogenous genomic copy of the corresponding gene. An AS polynucleotide is a polynucleotide which comprises consecutive nucleotides having a sequence of sufficient length and homology to a sequence present within the sequence of the target gene to permit hybridization of the AS to the gene. Many reviews have covered the main aspects of antisense (AS) technology and its enormous therapeutic potential (Aboul-Fadl T., Curr Med Chem. 2005; 12(19):2193-214; Crooke S T, Curr Mol Med. 2004 August; 4(5):465-87; Crooke S T, Annu Rev Med. 2004; 55:61-95; Vacek M et al., Cell Mol Life Sci. 2003 May; 60(5):825-33; Cho-Chung Y S, Arch Pharm Res. 2003 March; 26(3):183-91. There are further reviews on the chemical (Crooke, 1995; Uhlmann et al, 1990), cellular (Wagner, 1994) and therapeutic (Hanania, et al, 1995; Scanlon, et al, 1995; Gewirtz, 1993) aspects of this technology. Antisense intervention in the expression of specific genes can be achieved by the use of synthetic AS oligonucleotide sequences (for recent reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal, 1996; LevLehman et al, 1997).
AS oligonucleotide sequences may be short sequences of DNA, typically 15-30 mer but may be as small as 7 mer (Wagner et al, 1996), designed to complement a target mRNA of interest and form an RNA:AS duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA. Moreover, certain AS nucleotide sequences can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation (Calabretta et al, 1996 Semin Oncol. 23(1):78-87). In that case, RNase H will cleave the RNA component of the duplex and can potentially release the AS to further hybridize with additional molecules of the target RNA. An additional mode of action results from the interaction of AS with genomic DNA to form a triple helix which can be transcriptionally inactive.
The sequence target segment for the antisense oligonucleotide is selected such that the sequence exhibits suitable energy related characteristics important for oligonucleotide duplex formation with their complementary templates, and shows a low potential for self-dimerization or self-complementation [Anazodo et al., 19961. For example, the computer program OLIGO (Primer Analysis Software, Version 3.4), can be used to determine antisense sequence melting temperature, free energy properties, and to estimate potential self-dimer formation and self-complimentary properties. The program allows the determination of a qualitative estimation of these two parameters (potential self-dimer formation and self-complimentary) and provides an indication of “no potential” or “some potential” or “essentially complete potential”. Using this program target segments are generally selected that have estimates of no potential in these parameters. However, segments can be used that have “some potential” in one of the categories. A balance of the parameters is used in the selection as is known in the art. Further, the oligonucleotides are also selected as needed so that analogue substitution do not substantially affect function.
Phosphorothioate antisense oligonucleotides do not normally show significant toxicity at concentrations that are effective and exhibit sufficient pharmacodynamic half-lives in animals (Agarwal et al., 1996) and are nuclease resistant. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP), for the establishment of tectal plate formation in chick (Galileo et al., 1991) and for the N-myc protein, responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (epithelial vs. neuroblastic cells, which differ in their colony forming abilities, tumorigenicity and adherence) (Rosolen et al., 1990; Whitesell et al, 1991). Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), having mitogenic and angiogenic properties, suppressed 80% of growth in glioma cells (Morrison, 1991) in a saturable and specific manner. Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes (Akhter et al., 1991). Following their interaction with the cellular plasma membrane, they are actively (or passively) transported into living cells (Loke et al., 1989), in a saturable mechanism predicted to involve specific receptors (Yakubov et al., 1989).
Ribozymes
A “ribozyme” is an RNA molecule that possesses RNA catalytic ability (see Cech for review) and cleaves a specific site in a target RNA. In accordance with the present invention, ribozymes which cleave mRNA may be utilized as inhibitors. This may be necessary in cases where antisense therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then be used that will target the a gene associated with a bone marrow disease. The number of RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stochiochemistry. (Hampel and Tritz, 1989; Uhlenbeck, 1987).
Ribozymes catalyze the phosphodiester bond cleavage of RNA. Several ribozyme structural families have been identified including Group I introns, RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Pat. No. 5,225,347). The latter two families are derived from viroids and virusoids, in which the ribozyme is believed to separate monomers from oligomers created during rolling circle replication (Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994). In general the ribozyme has a length of from about 30-100 nucleotides. Delivery of ribozymes is similar to that of AS fragments and/or siRNA molecules.
Screening of Inactivation Compounds for Nrf2:
Some of the compounds and compositions of the present invention may be used in a screening assay for identifying and isolating compounds that modulate the activity of the NRF2 gene, in particular compounds that modulate a disorder accompanied by an elevated level of NRF2. The compounds to be screened comprise inter alia substances such as small chemical molecules and antisense oligonucleotides.
The inhibitory activity of the compounds of the present invention on Nrf2 expression may be used to determine the interaction of an additional compound with the target polypeptide, e.g., if the additional compound competes with the oligonucleotides of the present invention for inhibition of Nrf2, or if the additional compound rescues said inhibition. The inhibition or activation can be tested by various means, such as, inter alia, assaying for the product of the activity of the Nrf2 polypeptide or displacement of binding compound from the Nrf2 polypeptide in radioactive or fluorescent competition assays.
The present invention is illustrated in detail below with reference to the Examples, but is not to be construed as being limited thereto.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
EXAMPLES
General Methods in Molecular Biology
Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and as in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and as in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, Calif. (1990). In situ (In cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al., 1996, Blood 87:3822.) Methods of performing RT-PCR are also well known in the art.
Example 1
Generation of Sequences for Active siRNA Compounds
Using proprietary algorithms and the known sequence of Nrf2 (gi2014957), the sequences of many potential siRNAs were generated. Table A shows a list of 19-mers siRNAs specific to Nrf2 which are either human-specific, or human and cross-species with other species. Table B shows a list of 21-mers siRNAs specific to Nrf2 which are either human-specific, or human and cross-species with other species. All siRNAs are depicted in 5′ to 3′ orientation, and the sense and complementary antisense sequences are depicted on the same line in the Table. The sense siRNAs in Table A have SEQ ID NOS: 1-503 and the antisense siRNAs in Table A have SEQ ID NOS: 504-1006. The sense siRNAs in Table B have SEQ ID NOS: 1007-1506 and the antisense siRNAs in Table B have SEQ ID NOS: 1507-2006.







TABLE A







19-mers siRNAs specific to Nrf2 (SEQ ID NOS: 1-1006)











SEQ ID NO.
Sense siRNA
SEQ ID NO.
Antisense siRNA
Human-20149575














1
GGGGUAAGAAUAAAGUGGC
504
GCCACUUUAUUCUUACCCC
[1624-1642](19/19)


 


2
GGAGGGGUAAGAAUAAAGU
505
ACUUUAUUCUUACCCCUCC
[1621-1639](19/19)


 


3
GCAAAACUAACCACUAUGU
506
ACAUAGUGGUUAGUUUUGC
[2270-2288](19/19)


 


4
AAGGAGAAAAUGACAAAAG
507
CUUUUGUCAUUUUCUCCUU
[1741-1759](19/19)


 


5
CAGAAUUGCAGAAAAAGAA
508
UUCUUUUUCUGCAAUUCUG
[1647-1665](19/19)


 


6
GGGUAAGAAUAAAGUGGCU
509
AGCCACUUUAUUCUUACCC
[1625-1643](19/19)


 


7
GCCCUCACCUGCUACUUUA
510
UAAAGUAGCAGGUGAGGGC
[1010-1028](19/19)


 


8
AGGGGUAAGAAUAAAGUGG
511
CCACUUUAUUCUUACCCCU
[1623-1641](19/19)


 


9
AGGAGGGGUAAGAAUAAAG
512
CUUUAUUCUUACCCCUCCU
[1620-1638](19/19)


 


10
UCCCGUUUGUAGAUGACAA
513
UUGUCAUCUACAAACGGGA
[493-511](19/19)


 


11
AGGAGAAAAUGACAAAAGC
514
GCUUUUGUCAUUUUCUCCU
[1742-1760](19/19)


 


12
GCUCAGAAUUGCAGAAAAA
515
UUUUUCUGCAAUUCUGAGC
[1644-1662](19/19)


 


13
GAGAAAGAAUUGCCUGUAA
516
UUACAGGCAAUUCUUUCUC
[1392-1410](19/19)


 


14
UCCGGCAUUUCACUAAACA
517
UGUUUAGUGAAAUGCCGGA
[1143-1161](19/19)


 


15
GAGUUACAGUGUCUUAAUA
518
UAUUAAGACACUGUAACUC
[699-717](19/19)


 


16
GCCAAAACUAGUAUAGAAA
519
UUUCUAUACUAGUUUUGGC
[2044-2062](19/19)


 


17
AGCCAGAUGUUAAGAAAAA
520
UUUUUCUUAACAUCUGGCU
[1909-1927](19/19)


 


18
GAAGCCAGAUGUUAAGAAA
521
UUUCUUAACAUCUGGCUUC
[1907-1925](19/19)


 


19
AGAAGCCAGAUGUUAAGAA
522
UUCUUAACAUCUGGCUUCU
[1906-1924](19/19)


 


20
UCAGAAUUGCAGAAAAAGA
523
UCUUUUUCUGCAAUUCUGA
[1646-1664](19/19)


 


21
UAGGAGGGGUAAGAAUAAA
524
UUUAUUCUUACCCCUCCUA
[1619-1637](19/19)


 


22
CACUCUCUGAACUUCUAAA
525
UUUAGAAGUUCAGAGAGUG
[1036-1054](19/19)


 


23
UAGCCCCUGUUGAUUUAGA
526
UCUAAAUCAACAGGGGCUA
[628-646](19/19)


 


24
GAUCUGCCAACUACUCCCA
527
UGGGAGUAGUUGGCAGAUC
[406-424](19/19)


 


25
GCAAAAUCAUAGCCAAAAC
528
GUUUUGGCUAUGAUUUUGC
[2033-2051](19/19)


 


26
GUAAGAAGCCAGAUGUUAA
529
UUAACAUCUGGCUUCUUAC
[1903-1921](19/19)


 


27
UAAAGUGGCUGCUCAGAAU
530
AUUCUGAGCAGCCACUUUA
[1634-1652](19/19)


 


28
AGAAUAAAGUGGCUGCUCA
531
UGAGCAGCCACUUUAUUCU
[1630-1648](19/19)


 


29
AGGAAAGACAAGAACAACU
532
AGUUGUUCUUGUCUUUCCU
[280-298](19/19)


 


30
AAAGGAAAGACAAGAACAA
533
UUGUUCUUGUCUUUCCUUU
[278-296](19/19)


 


31
AACUUGAAAAGGAAAGACA
534
UGUCUUUCCUUUUCAAGUU
[271-289](19/19)


 


32
GACAAACAUUCAAGCCGCU
535
AGCGGCUUGAAUGUUUGUC
[1443-1461](19/19)


 


33
GACUCCGGCAUUUCACUAA
536
UUAGUGAAAUGCCGGAGUC
[1140-1158](19/19)


 


34
ACCAAAACCACCCUGAAAG
537
CUUUCAGGGUGGUUUUGGU
[1099-1117](19/19)


 


35
ACUCUCUGAACUUCUAAAU
538
AUUUAGAAGUUCAGAGAGU
[1037-1055](19/19)


 


36
GACAGUGAACUCAUUAAAU
539
AUUUAAUGAGUUCACUGUC
[911-929](19/19)


 


37
AAGUUUGGGAGGAGCUAUU
540
AAUAGCUCCUCCCAAACUU
[670-688](19/19)


 


38
UUGUAGAUGACAAUGAGGU
541
ACCUCAUUGUCAUCUACAA
[499-517](19/19)


 


39
AAACUAACCACUAUGUACU
542
AGUACAUAGUGGUUAGUUU
[2273-2291](19/19)


 


40
CAAAAUCAUAGCCAAAACU
543
AGUUUUGGCUAUGAUUUUG
[2034-2052](19/19)


 


41
UAAGAAGCCAGAUGUUAAG
544
CUUAACAUCUGGCUUCUUA
[1904-1922](19/19)


 


42
ACGUGAUGAAGAUGGAAAA
545
UUUUCCAUCUUCAUCACGU
[1817-1835](19/19)


 


43
CUGGAAAAUAUAGUAGAAC
546
GUUCUACUAUAUUUUCCAG
[1668-1686](19/19)


 


44
GAAAAGGAAAGACAAGAAC
547
GUUCUUGUCUUUCCUUUUC
[276-294](19/19)


 


45
UGAAAAGGAAAGACAAGAA
548
UUCUUGUCUUUCCUUUUCA
[275-293](19/19)


 


46
UGGAGACACACUACUUGGC
549
GCCAAGUAGUGUGUCUCCA
[1211-1229](19/19)


 


47
UCAACCAAAACCACCCUGA
550
UCAGGGUGGUUUUGGUUGA
[1096-1114](19/19)


 


48
CCCUCACCUGCUACUUUAA
551
UUAAAGUAGCAGGUGAGGG
[1011-1029](19/19)


 


49
UGACAGUGAACUCAUUAAA
552
UUUAAUGAGUUCACUGUCA
[910-928](19/19)


 


50
UUGGAGGCAAGAUAUAGAU
553
AUCUAUAUCUUGCCUCCAA
[182-200](19/19)


 


51
GAGCUAUUAUCCAUUCCUG
554
CAGGAAUGGAUAAUAGCUC
[681-699](19/19)


 


52
UAGAUGACAAUGAGGUUUC
555
GAAACCUCAUUGUCAUCUA
[502-520](19/19)


 


53
UGCCCACAUUCCCAAAUCA
556
UGAUUUGGGAAUGUGGGCA
[428-446](19/19)


 


54
AGCCAAAACUAGUAUAGAA
557
UUCUAUACUAGUUUUGGCU
[2043-2061](19/19)


 


55
AAAGUAAGAAGCCAGAUGU
558
ACAUCUGGCUUCUUACUUU
[1900-1918](19/19)


 


56
CCAAAAGUAAGAAGCCAGA
559
UCUGGCUUCUUACUUUUGG
[1897-1915](19/19)


 


57
CCCAAAAGUAAGAAGCCAG
560
CUGGCUUCUUACUUUUGGG
[1896-1914](19/19)


 


58
UACGUGAUGAAGAUGGAAA
561
UUUCCAUCUUCAUCACGUA
[1816-1834](19/19)


 


59
GCUCAAAGAAAAAGGAGAA
562
UUCUCCUUUUUCUUUGAGC
[1730-1748](19/19)


 


60
AACUAGAGCAAGAUUUAGA
563
UCUAAAUCUUGCUCUAGUU
[1684-1702](19/19)


 


61
AAGUGGCUGCUCAGAAUUG
564
CAAUUCUGAGCAGCCACUU
[1636-1654](19/19)


 


62
GUAGGAGGGGUAAGAAUAA
565
UUAUUCUUACCCCUCCUAC
[1618-1636](19/19)


 


63
UCAACUUGCAUUAAUUCGG
566
CCGAAUUAAUGCAAGUUGA
[1592-1610](19/19)


 


64
AAAGACAAGAACAACUCCA
567
UGGAGUUGUUCUUGUCUUU
[283-301](19/19)


 


65
AGAAAGAAUUGCCUGUAAG
568
CUUACAGGCAAUUCUUUCU
[1393-1411](19/19)


 


66
ACACACCAGAGAAAGAAUU
569
AAUUCUUUCUCUGGUGUGU
[1384-1402](19/19)


 


67
AACACCAGUACAUUCUUCU
570
AGAAGAAUGUACUGGUGUU
[1295-1313](19/19)


 


68
AAACACCAGUACAUUCUUC
571
GAAGAAUGUACUGGUGUUU
[1294-1312](19/19)


 


69
ACAGAAUGGUCCUAAAACA
572
UGUUUUAGGACCAUUCUGU
[1280-1298](19/19)


 


70
AAACCACCCUGAAAGCACA
573
UGUGCUUUCAGGGUGGUUU
[1103-1121](19/19)


 


71
AACCAAAACCACCCUGAAA
574
UUUCAGGGUGGUUUUGGUU
[1098-1116](19/19)


 


72
UCUCUGAACUUCUAAAUGG
575
CCAUUUAGAAGUUCAGAGA
[1039-1057](19/19)


 


73
UUCACUCUCUGAACUUCUA
576
UAGAAGUUCAGAGAGUGAA
[1034-1052](19/19)


 


74
GUAGUCCACAUUUUCUUAA
577
UUAAGAAAAUGUGGACUAC
[838-856](19/19)


 


75
UUGAAAAUGACAAGCUGGU
578
ACCAGCUUGUCAUUUUCAA
[718-736](19/19)


 


76
CAGUCUUCAUUGCUACUAA
579
UUAGUAGCAAUGAAGACUG
[574-592](19/19)


 


77
AUGACAAUGAGGUUUCUUC
580
GAAGAAACCUCAUUGUCAU
[505-523](19/19)


 


78
AGAUGACAAUGAGGUUUCU
581
AGAAACCUCAUUGUCAUCU
[503-521](19/19)


 


79
GUAGAUGACAAUGAGGUUU
582
AAACCUCAUUGUCAUCUAC
[501-519](19/19)


 


80
UGCCAACUACUCCCAGGUU
583
AACCUGGGAGUAGUUGGCA
[410-428](19/19)


 


81
AAGCCAGAUGUUAAGAAAA
584
UUUUCUUAACAUCUGGCUU
[1908-1926](19/19)


 


82
ACCUUAUUCUCCUAGUGAA
585
UUCACUAGGAGAAUAAGGU
[1835-1853](19/19)


 


83
ACCUACUGAAAAAACAACU
586
AGUUGUUUUUUCAGUAGGU
[1765-1783](19/19)


 


84
ACAAAAGCCUUCACCUACU
587
AGUAGGUGAAGGCUUUUGU
[1753-1771](19/19)


 


85
UGCUCAAAGAAAAAGGAGA
588
UCUCCUUUUUCUUUGAGCA
[1729-1747](19/19)


 


86
CAACUUGCAUUAAUUCGGG
589
CCCGAAUUAAUGCAAGUUG
[1593-1611](19/19)


 


87
GGAAAGACAAGAACAACUC
590
GAGUUGUUCUUGUCUUUCC
[281-299](19/19)


 


88
UUGAAAAGGAAAGACAAGA
591
UCUUGUCUUUCCUUUUCAA
[274-292](19/19)


 


89
CAAAAGACAAACAUUCAAG
592
CUUGAAUGUUUGUCUUUUG
[1438-1456](19/19)


 


90
UCACAAAAGACAAACAUUC
593
GAAUGUUUGUCUUUUGUGA
[1435-1453](19/19)


 


91
ACCCCAUUCACAAAAGACA
594
UGUCUUUUGUGAAUGGGGU
[1428-1446](19/19)


 


92
AGAAUUGCCUGUAAGUCCU
595
AGGACUUACAGGCAAUUCU
[1397-1415](19/19)


 


93
AGCGACGGAAAGAGUAUGA
596
UCAUACUCUUUCCGUCGCU
[235-253](19/19)


 


94
AAGCACAGCAGAAUUCAAU
597
AUUGAAUUCUGCUGUGCUU
[1115-1133](19/19)


 


95
CUCUGAACUUCUAAAUGGG
598
CCCAUUUAGAAGUUCAGAG
[1040-1058](19/19)


 


96
UUGACAGUGAACUCAUUAA
599
UUAAUGAGUUCACUGUCAA
[909-927](19/19)


 


97
UAAGUCGAGAAGUAUUUGA
600
UCAAAUACUUCUCGACUUA
[208-226](19/19)


 


98
UGGAGUAAGUCGAGAAGUA
601
UACUUCUCGACUUACUCCA
[203-221](19/19)


 


99
UACUCAUCUAUACCCUCAA
602
UUGAGGGUAUAGAUGAGUA
[798-816](19/19)


 


100
UCCAAGUCCAGAAGCCAAA
603
UUUGGCUUCUGGACUUGGA
[752-770](19/19)


 


101
CUGUUGAUUUAGACGGUAU
604
AUACCGUCUAAAUCAACAG
[634-652](19/19)


 


102
UUGCUACUAAUCAGGCUCA
605
UGAGCCUGAUUAGUAGCAA
[583-601](19/19)


 


103
GCCCAGUCUUCAUUGCUAC
606
GUAGCAAUGAAGACUGGGC
[571-589](19/19)


 


104
CAUUCCCGUUUGUAGAUGA
607
UCAUCUACAAACGGGAAUG
[490-508](19/19)


 


105
AUGCUUUGUACUUUGAUGA
608
UCAUCAAAGUACAAAGCAU
[448-466](19/19)


 


106
CAGAUGCUUUGUACUUUGA
609
UCAAAGUACAAAGCAUCUG
[445-463](19/19)


 


107
AAGAGCUGGUACUAAUAAA
610
UUUAUUAGUACCAGCUCUU
[2370-2388](19/19)


 


108
GGACAAAAAAUGGCAUUUU
611
AAAAUGCCAUUUUUUGUCC
[2310-2328](19/19)


 


109
GUGAAAUGCUCAUACUUUA
612
UAAAGUAUGAGCAUUUCAC
[2001-2019](19/19)


 


110
AACCUUAUUCUCCUAGUGA
613
UCACUAGGAGAAUAAGGUU
[1834-1852](19/19)


 


111
AAAACCUUAUUCUCCUAGU
614
ACUAGGAGAAUAAGGUUUU
[1832-1850](19/19)


 


112
AAACAACUCAGCACCUUAU
615
AUAAGGUGCUGAGUUGUUU
[1776-1794](19/19)


 


113
CACCUACUGAAAAAACAAC
616
GUUGUUUUUUCAGUAGGUG
[1764-1782](19/19)


 


114
UUCACCUACUGAAAAAACA
617
UGUUUUUUCAGUAGGUGAA
[1762-1780](19/19)


 


115
ACAAGAACAACUCCAAAAG
618
CUUUUGGAGUUGUUCUUGU
[287-305](19/19)


 


116
UUGCAUUAAUUCGGGAUAU
619
AUAUCCCGAAUUAAUGCAA
[1597-1615](19/19)


 


117
AACUUGCAUUAAUUCGGGA
620
UCCCGAAUUAAUGCAAGUU
[1594-1612](19/19)


 


118
CACAAAAGACAAACAUUCA
621
UGAAUGUUUGUCUUUUGUG
[1436-1454](19/19)


 


119
CCCAUUCACAAAAGACAAA
622
UUUGUCUUUUGUGAAUGGG
[1430-1448](19/19)


 


120
GAACACACCAGAGAAAGAA
623
UUCUUUCUCUGGUGUGUUC
[1382-1400](19/19)


 


121
ACGGUCCACAGCUCAUCAU
624
AUGAUGAGCUGUGGACCGU
[97-115](19/19)


 


122
UGAGAACACACCAGAGAAA
625
UUUCUCUGGUGUGUUCUCA
[1379-1397](19/19)


 


123
GUGAUUCUGAAGUGGAAGA
626
UCUUCCACUUCAGAAUCAC
[1234-1252](19/19)


 


124
ACAGCAGAAUUCAAUGAUU
627
AAUCAUUGAAUUCUGCUGU
[1119-1137](19/19)


 


125
GCACAGCAGAAUUCAAUGA
628
UCAUUGAAUUCUGCUGUGC
[1117-1135](19/19)


 


126
CUCAUUAAAUUCAGAUGCC
629
GGCAUCUGAAUUUAAUGAG
[920-938](19/19)


 


127
ACUCAUUAAAUUCAGAUGC
630
GCAUCUGAAUUUAAUGAGU
[919-937](19/19)


 


128
AACCAGUUGACAGUGAACU
631
AGUUCACUGUCAACUGGUU
[903-921](19/19)


 


129
UCCUUCAGCAGCAUCCUCU
632
AGAGGAUGCUGCUGAAGGA
[870-888](19/19)


 


130
GAGGAUUCCUUCAGCAGCA
633
UGCUGCUGAAGGAAUCCUC
[864-882](19/19)


 


131
AGUAAGUCGAGAAGUAUUU
634
AAAUACUUCUCGACUUACU
[206-224](19/19)


 


132
GAGUAAGUCGAGAAGUAUU
635
AAUACUUCUCGACUUACUC
[205-223](19/19)


 


133
ACUCAUCUAUACCCUCAAU
636
AUUGAGGGUAUAGAUGAGU
[799-817](19/19)


 


134
UUACUCAUCUAUACCCUCA
637
UGAGGGUAUAGAUGAGUAA
[797-815](19/19)


 


135
UGAGUUACAGUGUCUUAAU
638
AUUAAGACACUGUAACUCA
[698-716](19/19)


 


136
UUUGGAGGCAAGAUAUAGA
639
UCUAUAUCUUGCCUCCAAA
[181-199](19/19)


 


137
AGGACAUUGAGCAAGUUUG
640
CAAACUUGCUCAAUGUCCU
[658-676](19/19)


 


138
UAGACGGUAUGCAACAGGA
641
UCCUGUUGCAUACCGUCUA
[643-661](19/19)


 


139
UUGAUUUAGACGGUAUGCA
642
UGCAUACCGUCUAAAUCAA
[637-655](19/19)


 


140
UUCAUUGCUACUAAUCAGG
643
CCUGAUUAGUAGCAAUGAA
[579-597](19/19)


 


141
AGUCUUCAUUGCUACUAAU
644
AUUAGUAGCAAUGAAGACU
[575-593](19/19)


 


142
CGGCUACGUUUCAGUCACU
645
AGUGACUGAAACGUAGCCG
[523-541](19/19)


 


143
AGGACAUGGAUUUGAUUGA
646
UCAAUCAAAUCCAUGUCCU
[157-175](19/19)


 


144
GCCCACAUUCCCAAAUCAG
647
CUGAUUUGGGAAUGUGGGC
[429-447](19/19)


 


145
AGCAGGACAUGGAUUUGAU
648
AUCAAAUCCAUGUCCUGCU
[154-172](19/19)


 


146
AGCUGGUACUAAUAAAGGA
649
UCCUUUAUUAGUACCAGCU
[2373-2391](19/19)


 


147
GAGCUGGUACUAAUAAAGG
650
CCUUUAUUAGUACCAGCUC
[2372-2390](19/19)


 


148
AGAGCUGGUACUAAUAAAG
651
CUUUAUUAGUACCAGCUCU
[2371-2389](19/19)


 


149
CUAACCACUAUGUACUUUU
652
AAAAGUACAUAGUGGUUAG
[2276-2294](19/19)


 


150
ACAUCUGGCUAAAAAGAAA
653
UUUCUUUUUAGCCAGAUGU
[2246-2264](19/19)


 


151
CUGUUCUUAUGUCAUUUGU
654
ACAAAUGACAUAAGAACAG
[2199-2217](19/19)


 


152
UGGGCUAGUUUCUGUGUAA
655
UUACACAGAAACUAGCCCA
[2152-2170](19/19)


 


153
GUAAACAAUUUCUUAGGAC
656
GUCCUAAGAAAUUGUUUAC
[2127-2145](19/19)


 


154
CUGUAAACAAUUUCUUAGG
657
CCUAAGAAAUUGUUUACAG
[2125-2143](19/19)


 


155
GUCAGUAUGUUGAAUCAGU
658
ACUGAUUCAACAUACUGAC
[2093-2111](19/19)


 


156
GGCAAUGUUUUCCUUGUUC
659
GAACAAGGAAAACAUUGCC
[1878-1896](19/19)


 


157
GCACCUUAUAUCUCGAAGU
660
ACUUCGAGAUAUAAGGUGC
[1786-1804](19/19)


 


158
GAAAAAACAACUCAGCACC
661
GGUGCUGAGUUGUUUUUUC
[1772-1790](19/19)


 


159
AGAGCAAGAUUUAGAUCAU
662
AUGAUCUAAAUCUUGCUCU
[1688-1706](19/19)


 


160
UAGAGCAAGAUUUAGAUCA
663
UGAUCUAAAUCUUGCUCUA
[1687-1705](19/19)


 


161
UAGAACUAGAGCAAGAUUU
664
AAAUCUUGCUCUAGUUCUA
[1681-1699](19/19)


 


162
ACGUAGGAGGGGUAAGAAU
665
AUUCUUACCCCUCCUACGU
[1616-1634](19/19)


 


163
UCCCUGUUGUUGACUUCAA
666
UUGAAGUCAACAACAGGGA
[1540-1558](19/19)


 


164
UCCCUGUAGAAAAAAUCAU
667
AUGAUUUUUUCUACAGGGA
[1516-1534](19/19)


 


165
UCACAAGAGAUGAACUUAG
668
CUAAGUUCAUCUCUUGUGA
[1474-1492](19/19)


 


166
AGAGAAAGAAUUGCCUGUA
669
UACAGGCAAUUCUUUCUCU
[1391-1409](19/19)


 


167
ACCAGAGAAAGAAUUGCCU
670
AGGCAAUUCUUUCUCUGGU
[1388-1406](19/19)


 


168
CACCAGAGAAAGAAUUGCC
671
GGCAAUUCUUUCUCUGGUG
[1387-1405](19/19)


 


169
AGUAUGAGCUGGAAAAACA
672
UGUUUUUCCAGCUCAUACU
[247-265](19/19)


 


170
GUACAUUCUUCUGGGGAUA
673
UAUCCCCAGAAGAAUGUAC
[1302-1320](19/19)


 


171
CAAACAGAAUGGUCCUAAA
674
UUUAGGACCAUUCUGUUUG
[1277-1295](19/19)


 


172
CUGGAAGUGUCAAACAGAA
675
UUCUGUUUGACACUUCCAG
[1267-1285](19/19)


 


173
GGAAAGAGUAUGAGCUGGA
676
UCCAGCUCAUACUCUUUCC
[241-259](19/19)


 


174
CAGUGAUUCUGAAGUGGAA
677
UUCCACUUCAGAAUCACUG
[1232-1250](19/19)


 


175
GACGGAAAGAGUAUGAGCU
678
AGCUCAUACUCUUUCCGUC
[238-256](19/19)


 


176
CACAGCAGAAUUCAAUGAU
679
AUCAUUGAAUUCUGCUGUG
[1118-1136](19/19)


 


177
GCCACAGUCAACACAGAUU
680
AAUCUGUGUUGACUGUGGC
[936-954](19/19)


 


178
UAAAUUCAGAUGCCACAGU
681
ACUGUGGCAUCUGAAUUUA
[925-943](19/19)


 


179
CCAGUUGACAGUGAACUCA
682
UGAGUUCACUGUCAACUGG
[905-923](19/19)


 


180
GUCGAGAAGUAUUUGACUU
683
AAGUCAAAUACUUCUCGAC
[211-229](19/19)


 


181
CUGUAGUCCACAUUUUCUU
684
AAGAAAAUGUGGACUACAG
[836-854](19/19)


 


182
GGAGUAAGUCGAGAAGUAU
685
AUACUUCUCGACUUACUCC
[204-222](19/19)


 


183
GACAGAAGUUGACAAUUAU
686
AUAAUUGUCAACUUCUGUC
[773-791](19/19)


 


184
GAGACUACCAUGGUUCCAA
687
UUGGAACCAUGGUAGUCUC
[738-756](19/19)


 


185
CUAUUAUCCAUUCCUGAGU
688
ACUCAGGAAUGGAUAAUAG
[684-702](19/19)


 


186
GACGGUAUGCAACAGGACA
689
UGUCCUGUUGCAUACCGUC
[645-663](19/19)


 


187
CGAGAGCCCAGUCUUCAUU
690
AAUGAAGACUGGGCUCUCG
[566-584](19/19)


 


188
UUGAUUGACAUACUUUGGA
691
UCCAAAGUAUGUCAAUCAA
[168-186](19/19)


 


189
GUCACUUGUUCCUGAUAUU
692
AAUAUCAGGAACAAGUGAC
[536-554](19/19)


 


190
UCGGCUACGUUUCAGUCAC
693
GUGACUGAAACGUAGCCGA
[522-540](19/19)


 


191
GAGGUUUCUUCGGCUACGU
694
ACGUAGCCGAAGAAACCUC
[513-531](19/19)


 


192
GACAUUCCCGUUUGUAGAU
695
AUCUACAAACGGGAAUGUC
[488-506](19/19)


 


193
GACAUGGAUUUGAUUGACA
696
UGUCAAUCAAAUCCAUGUC
[159-177](19/19)


 


194
UCAGAUGCUUUGUACUUUG
697
CAAAGUACAAAGCAUCUGA
[444-462](19/19)


 


195
CAGCAGGACAUGGAUUUGA
698
UCAAAUCCAUGUCCUGCUG
[153-171](19/19)


 


196
AUGGACAAAAAAUGGCAUU
699
AAUGCCAUUUUUUGUCCAU
[2308-2326](19/19)


 


197
GUAUGGACAAAAAAUGGCA
700
UGCCAUUUUUUGUCCAUAC
[2306-2324](19/19)


 


198
CAUCUGGCUAAAAAGAAAU
701
AUUUCUUUUUAGCCAGAUG
[2247-2265](19/19)


 


199
GACAUCUGGCUAAAAAGAA
702
UUCUUUUUAGCCAGAUGUC
[2245-2263](19/19)


 


200
GAGCUAGUUUUUUUGUACU
703
AGUACAAAAAAACUAGCUC
[1956-1974](19/19)


 


201
GAAGAUGGAAAACCUUAUU
704
AAUAAGGUUUUCCAUCUUC
[1824-1842](19/19)


 


202
GCUACGUGAUGAAGAUGGA
705
UCCAUCUUCAUCACGUAGC
[1814-1832](19/19)


 


203
UGCUAGGUGAUGAAGAUGG
706
CCAUCUUCAUCACGUAGCA
[1813-1831](19/19)


 


204
GAGCAAGAUUUAGAUCAUU
707
AAUGAUCUAAAUCUUGCUC
[1689-1707](19/19)


 


205
ACUAGAGCAAGAUUUAGAU
708
AUCUAAAUCUUGCUCUAGU
[1685-1703](19/19)


 


206
UAGUAGAACUAGAGCAAGA
709
UCUUGCUCUAGUUCUACUA
[1678-1696](19/19)


 


207
AUAUCCCAUUCCCUGUAGA
710
UCUACAGGGAAUGGGAUAU
[1507-1525](19/19)


 


208
CCAUAUCCCAUUCCCUGUA
711
UACAGGGAAUGGGAUAUGG
[1505-1523](19/19)


 


209
CCCCAUUCACAAAAGACAA
712
UUGUCUUUUGUGAAUGGGG
[1429-1447](19/19)


 


210
ACACCAGAGAAAGAAUUGC
713
GCAAUUCUUUCUCUGGUGU
[1386-1404](19/19)


 


211
UCAAACAGAAUGGUCCUAA
714
UUAGGACCAUUCUGUUUGA
[1276-1294](19/19)


 


212
AAGAGUAUGAGCUGGAAAA
715
UUUUCCAGCUCAUACUCUU
[244-262](19/19)


 


213
UGCCCCUGGAAGUGUCAAA
716
UUUGACACUUCCAGGGGCA
[1262-1280](19/19)


 


214
GCGACGGAAAGAGUAUGAG
717
CUCAUACUCUUUCCGUCGC
[236-254](19/19)


 


215
CUGAACUUCUAAAUGGGCC
718
GGCCCAUUUAGAAGUUCAG
[1042-1060](19/19)


 


216
CCACAGUCAACACAGAUUU
719
AAAUCUGUGUUGACUGUGG
[937-955](19/19)


 


217
AUGCUUUUGAGGAUUCCUU
720
AAGGAAUCCUCAAAAGCAU
[856-874](19/19)


 


218
CUCAAUGGAAAAAGAAGUA
721
UACUUCUUUUUCCAUUGAG
[812-830](19/19)


 


219
CUCAUCUAUACCCUCAAUG
722
CAUUGAGGGUAUAGAUGAG
[800-818](19/19)


 


220
ACAUUGAGCAAGUUUGGGA
723
UCCCAAACUUGCUCAAUGU
[661-679](19/19)


 


221
AACAGGACAUUGAGCAAGU
724
ACUUGCUCAAUGUCCUGUU
[655-673](19/19)


 


222
UUAGACGGUAUGCAACAGG
725
CCUGUUGCAUACCGUCUAA
[642-660](19/19)


 


223
ACCUGAAACUUCUGUUGCU
726
AGCAACAGAAGUUUCAGGU
[605-623](19/19)


 


224
CCAGUCUUCAUUGCUACUA
727
UAGUAGCAAUGAAGACUGG
[573-591](19/19)


 


225
AGAGCCCAGUCUUCAUUGC
728
GCAAUGAAGACUGGGCUCU
[568-586](19/19)


 


226
AGUCACUUGUUCCUGAUAU
729
AUAUCAGGAACAAGUGACU
[535-553](19/19)


 


227
UGGAUUUGAUUGACAUACU
730
AGUAUGUCAAUCAAAUCCA
[163-181](19/19)


 


228
CAAUGAGGUUUCUUCGGCU
731
AGCCGAAGAAACCUCAUUG
[509-527](19/19)


 


229
ACAAUGAGGUUUCUUCGGC
732
GCCGAAGAAACCUCAUUGU
[508-526](19/19)


 


230
AGACAUUCCCGUUUGUAGA
733
UCUACAAACGGGAAUGUCU
[487-505](19/19)


 


231
GCUUUGUACUUUGAUGACU
734
AGUCAUCAAAGUACAAAGC
[450-468](19/19)


 


232
CCCACAUUCCCAAAUCAGA
735
UCUGAUUUGGGAAUGUGGG
[430-448](19/19)


 


233
ACAACUAGAUGAAGAGACA
736
UGUCUCUUCAUCUAGUUGU
[335-353](19/19)


 


234
UACAACUAGAUGAAGAGAC
737
GUCUCUUCAUCUAGUUGUA
[334-352](19/19)


 


235
UUUAAGAGCUGGUACUAAU
738
AUUAGUACCAGCUCUUAAA
[2367-2385](19/19)


 


236
UGGACAAAAAAUGGCAUUU
739
AAAUGCCAUUUUUUGUCCA
[2309-2327](19/19)


 


237
GGGCUAGUUUCUGUGUAAG
740
CUUACACAGAAACUAGCCC
[2153-2171](19/19)


 


238
UUGGGCUAGUUUCUGUGUA
741
UACACAGAAACUAGCCCAA
[2151-2169](19/19)


 


239
GUGUCAGUAUGUUGAAUCA
742
UGAUUCAACAUACUGACAC
[2091-2109](19/19)


 


240
GAUGUGAAAUGCUCAUACU
743
AGUAUGAGCAUUUCACAUC
[1998-2016](19/19)


 


241
CUCCUACUGUGAUGUGAAA
744
UUUCACAUCACAGUAGGAG
[1988-2006](19/19)


 


242
AGAUUUAGGAGGAUUUGAC
745
GUCAAAUCCUCCUAAAUCU
[1930-1948](19/19)


 


243
GCAAUGUUUUCCUUGUUCC
746
GGAACAAGGAAAACAUUGC
[1879-1897](19/19)


 


244
AACAAGAGAUGGCAAUGUU
747
AACAUUGCCAUCUCUUGUU
[1868-1886](19/19)


 


245
GCAGCAAACAAGAGAUGGC
748
GCCAUCUCUUGUUUGCUGC
[1862-1880](19/19)


 


246
CUCCUAGUGAAUACUCCCU
749
AGGGAGUAUUCACUAGGAG
[1843-1861](19/19)


 


247
CAGCACCUUAUAUCUCGAA
750
UUCGAGAUAUAAGGUGCUG
[1784-1802](19/19)


 


248
UCACCUACUGAAAAAACAA
751
UUGUUUUUUCAGUAGGUGA
[1763-1781](19/19)


 


249
GCAAGAUUUAGAUCAUUUG
752
CAAAUGAUCUAAAUCUUGC
[1691-1709](19/19)


 


250
CAGAAAAAGAAAACUGGAA
753
UUCCAGUUUUCUUUUUCUG
[1655-1673](19/19)


 


251
AUACGUAGGAGGGGUAAGA
754
UCUUACCCCUCCUACGUAU
[1614-1632](19/19)


 


252
UGCAUUAAUUCGGGAUAUA
755
UAUAUCCCGAAUUAAUGCA
[1598-1616](19/19)


 


253
AAGCUCUCCAUAUCCCAUU
756
AAUGGGAUAUGGAGAGCUU
[1498-1516](19/19)


 


254
CAUUCACAAAAGACAAACA
757
UGUUUGUCUUUUGUGAAUG
[1432-1450](19/19)


 


255
GAGUAUGAGCUGGAAAAAC
758
GUUUUUCCAGCUCAUACUC
[246-264](19/19)


 


256
CAGAAUGGUCCUAAAACAC
759
GUGUUUUAGGACCAUUCUG
[1281-1299](19/19)


 


257
GUGUCAAACAGAAUGGUCC
760
GGACCAUUCUGUUUGACAC
[1273-1291](19/19)


 


258
GAAGUGUCAAACAGAAUGG
761
CCAUUCUGUUUGACACUUC
[1270-1288](19/19)


 


259
CUGAAGUGGAAGAGCUAGA
762
UCUAGCUCUUCCACUUCAG
[1240-1258](19/19)


 


260
CCAGAACACUCAGUGGAAU
763
AUUCCACUGAGUGUUCUGG
[1182-1200](19/19)


 


261
UUCUGACUCCGGCAUUUCA
764
UGAAAUGCCGGAGUCAGAA
[1136-1154](19/19)


 


262
AUGAUUCUGACUCCGGCAU
765
AUGCCGGAGUCAGAAUCAU
[1132-1150](19/19)


 


263
GGCCCAUUGAUGUUUCUGA
766
UCAGAAACAUCAAUGGGCC
[1057-1075](19/19)


 


264
CACAGAUUUUGGUGAUGAA
767
UUCAUCACCAAAAUCUGUG
[947-965](19/19)


 


265
CAGUUGACAGUGAACUCAU
768
AUGAGUUCACUGUCAACUG
[906-924](19/19)


 


266
UUAAUGCUUUUGAGGAUUC
769
GAAUCCUCAAAAGCAUUAA
[853-871](19/19)


 


267
UCUUAAUGCUUUUGAGGAU
770
AUCCUCAAAAGCAUUAAGA
[851-869](19/19)


 


268
AGUCCACAUUUUCUUAAUG
771
CAUUAAGAAAAUGUGGACU
[840-858](19/19)


 


269
UGACAGAAGUUGACAAUUA
772
UAAUUGUCAACUUCUGUCA
[772-790](19/19)


 


270
CCAAACUGACAGAAGUUGA
773
UCAACUUCUGUCAGUUUGG
[766-784](19/19)


 


271
GUUGAGACUACCAUGGUUC
774
GAACCAUGGUAGUCUCAAC
[735-753](19/19)


 


272
ACAAGCUGGUUGAGACUAC
775
GUAGUCUCAACCAGCUUGU
[727-745](19/19)


 


273
AUACUUUGGAGGCAAGAUA
776
UAUCUUGCCUCCAAAGUAU
[177-195](19/19)


 


274
GACAUACUUUGGAGGCAAG
777
CUUGCCUCCAAAGUAUGUC
[174-192](19/19)


 


275
GUCACCUGAAACUUCUGUU
778
AACAGAAGUUUCAGGUGAC
[602-620](19/19)


 


276
UCGAGAGCCCAGUCUUCAU
779
AUGAAGACUGGGCUCUCGA
[565-583](19/19)


 


277
GUACUUUGAUGACUGCAUG
780
CAUGCAGUCAUCAAAGUAC
[455-473](19/19)


 


278
UGUACUUUGAUGACUGCAU
781
AUGCAGUCAUCAAAGUACA
[454-472](19/19)


 


279
GGACAUGGAUUUGAUUGAC
782
GUCAAUCAAAUCCAUGUCC
[158-176](19/19)


 


280
GAUGCUUUGUACUUUGAUG
783
CAUCAAAGUACAAAGCAUC
[447-465](19/19)


 


281
AUCAGAUGCUUUGUACUUU
784
AAAGUACAAAGCAUCUGAU
[443-461](19/19)


 


282
UUCCCAAAUCAGAUGCUUU
785
AAAGCAUCUGAUUUGGGAA
[436-454](19/19)


 


283
CAUUCCCAAAUCAGAUGCU
786
AGCAUCUGAUUUGGGAAUG
[434-452](19/19)


 


284
CCACAUUCCCAAAUCAGAU
787
AUCUGAUUUGGGAAUGUGG
[431-449](19/19)


 


285
AGUUACAACUAGAUGAAGA
788
UCUUCAUCUAGUUGUAACU
[331-349](19/19)


 


286
UAUGGACAAAAAAUGGCAU
789
AUGCCAUUUUUUGUCCAUA
[2307-2325](19/19)


 


287
ACUGUAUGGACAAAAAAUG
790
CAUUUUUUGUCCAUACAGU
[2303-2321](19/19)


 


288
UAUGACAUCUGGCUAAAAA
791
UUUUUAGCCAGAUGUCAUA
[2242-2260](19/19)


 


289
ACUGUUCUUAUGUCAUUUG
792
CAAAUGACAUAAGAACAGU
[2198-2216](19/19)


 


290
UAGUUUCUGUGUAAGUGUA
793
UACACUUACACAGAAACUA
[2157-2175](19/19)


 


291
GGCUAGUUUCUGUGUAAGU
794
ACUUACACAGAAACUAGCC
[2154-2172](19/19)


 


292
CAAUUUCUUAGGACACCAU
795
AUGGUGUCCUAAGAAAUUG
[2132-2150](19/19)


 


293
GUAUGUUGAAUCAGUAGUU
796
AACUACUGAUUCAACAUAC
[2097-2115](19/19)


 


294
UCAGUAUGUUGAAUCAGUA
797
UACUGAUUCAACAUACUGA
[2094-2112](19/19)


 


295
UGAUGUGAAAUGCUCAUAC
798
GUAUGAGCAUUUCACAUCA
[1997-2015](19/19)


 


296
AAAGCUCCUACUGUGAUGU
799
ACAUCACAGUAGGAGCUUU
[1984-2002](19/19)


 


297
UACUAAAAGCUCCUACUGU
800
ACAGUAGGAGCUUUUAGUA
[1979-1997](19/19)


 


298
GGAUUUGACCUUUUCUGAG
801
CUCAGAAAAGGUCAAAUCC
[1940-1958](19/19)


 


299
AGAGAUGGCAAUGUUUUCC
802
GGAAAACAUUGCCAUCUCU
[1872-1890](19/19)


 


300
AAACAAGAGAUGGCAAUGU
803
ACAUUGCCAUCUCUUGUUU
[1867-1885](19/19)


 


301
CAGCAAACAAGAGAUGGCA
804
UGCCAUCUCUUGUUUGCUG
[1863-1881](19/19)


 


302
CUAGUGAAUACUCCCUGCA
805
UGCAGGGAGUAUUCACUAG
[1846-1864](19/19)


 


303
AAGAUGGAAAACCUUAUUC
806
GAAUAAGGUUUUCCAUCUU
[1825-1843](19/19)


 


304
CAGCAUGCUACGUGAUGAA
807
UUCAUCACGUAGCAUGCUG
[1808-1826](19/19)


 


305
CACCUUAUAUCUCGAAGUU
808
AACUUCGAGAUAUAAGGUG
[1787-1805](19/19)


 


306
UCAGCACCUUAUAUCUCGA
809
UCGAGAUAUAAGGUGCUGA
[1783-1801](19/19)


 


307
AUAUAGUAGAACUAGAGCA
810
UGCUCUAGUUCUACUAUAU
[1675-1693](19/19)


 


308
UUGCAGAAAAAGAAAACUG
811
CAGUUUUCUUUUUCUGCAA
[1652-1670](19/19)


 


309
ACGAAAUGAUGUCCAAAGA
812
UCUUUGGACAUCAUUUCGU
[1558-1576](19/19)


 


310
ACCUCCCUGUUGUUGACUU
813
AAGUCAACAACAGGGAGGU
[1537-1555](19/19)


 


311
CCCUGUAGAAAAAAUCAUU
814
AAUGAUUUUUUCUACAGGG
[1517-1535](19/19)


 


312
GAGAUGAACUUAGGGCAAA
815
UUUGCCCUAAGUUCAUCUC
[1480-1498](19/19)


 


313
CACAAGAGAUGAACUUAGG
816
CCUAAGUUCAUCUCUUGUG
[1475-1493](19/19)


 


314
AACCCCAUUCACAAAAGAC
817
GUCUUUUGUGAAUGGGGUU
[1427-1445](19/19)


 


315
AGAACACACCAGAGAAAGA
818
UCUUUCUCUGGUGUGUUCU
[1381-1399](19/19)


 


316
GUCAAACAGAAUGGUCCUA
819
UAGGACCAUUCUGUUUGAC
[1275-1293](19/19)


 


317
GGAAGUGUCAAACAGAAUG
820
CAUUCUGUUUGACACUUCC
[1269-1287](19/19)


 


318
UGGAAGUGUCAAACAGAAU
821
AUUCUGUUUGACACUUCCA
[1268-1286](19/19)


 


319
CCCUGGAAGUGUCAAACAG
822
CUGUUUGACACUUCCAGGG
[1265-1283](19/19)


 


320
GUGGAAGAGCUAGAUAGUG
823
CACUAUCUAGCUCUUCCAC
[1245-1263](19/19)


 


321
ACCAGAACACUCAGUGGAA
824
UUCCACUGAGUGUUCUGGU
[1181-1199](19/19)


 


322
CAGCGACGGAAAGAGUAUG
825
CAUACUCUUUCCGUCGCUG
[234-252](19/19)


 


323
UGAUCUAUCACUUUGCAAA
826
UUUGCAAAGUGAUAGAUCA
[1073-1091](19/19)


 


324
GCCCAUUGAUGUUUCUGAU
827
AUCAGAAACAUCAAUGGGC
[1058-1076](19/19)


 


325
AGUCAACACAGAUUUUGGU
828
ACCAAAAUCUGUGUUGACU
[941-959](19/19)


 


326
AGUUGACAGUGAACUCAUU
829
AAUGAGUUCACUGUCAACU
[907-925](19/19)


 


327
ACUGUAGUCCACAUUUUCU
830
AGAAAAUGUGGACUACAGU
[835-853](19/19)


 


328
CAAUGGAAAAAGAAGUAGG
831
CCUACUUCUUUUUCCAUUG
[814-832](19/19)


 


329
CAAACUGACAGAAGUUGAC
832
GUCAACUUCUGUCAGUUUG
[767-785](19/19)


 


330
AGACUACCAUGGUUCCAAG
833
CUUGGAACCAUGGUAGUCU
[739-757](19/19)


 


331
ACAGGACAUUGAGCAAGUU
834
AACUUGCUCAAUGUCCUGU
[656-674](19/19)


 


332
UGACAUACUUUGGAGGCAA
835
UUGCCUCCAAAGUAUGUCA
[173-191](19/19)


 


333
ACGUUUCAGUCACUUGUUC
836
GAACAAGUGACUGAAACGU
[528-546](19/19)


 


334
CUACGUUUCAGUCACUUGU
837
ACAAGUGACUGAAACGUAG
[526-544](19/19)


 


335
GGCUACGUUUCAGUCACUU
838
AAGUGACUGAAACGUAGCC
[524-542](19/19)


 


336
GGAUUUGAUUGACAUACUU
839
AAGUAUGUCAAUCAAAUCC
[164-182](19/19)


 


337
UCUUCGGCUACGUUUCAGU
840
ACUGAAACGUAGCCGAAGA
[519-537](19/19)


 


338
CAGUCAGAAACCAGUGGAU
841
AUCCACUGGUUUCUGACUG
[390-408](19/19)


 


339
UGAAGAGACAGGUGAAUUU
842
AAAUUCACCUGUCUCUUCA
[344-362](19/19)


 


340
UAGAUGAAGAGACAGGUGA
843
UCACCUGUCUCUUCAUCUA
[340-358](19/19)


 


341
UAAGAGCUGGUACUAAUAA
844
UUAUUAGUACCAGCUCUUA
[2369-2387](19/19)


 


342
CUGUAUGGACAAAAAAUGG
845
CCAUUUUUUGUCCAUACAG
[2304-2322](19/19)


 


343
UGGAGUGUCAGUAUGUUGA
846
UCAACAUACUGACACUCCA
[2087-2105](19/19)


 


344
UGUGAAAUGCUCAUACUUU
847
AAAGUAUGAGCAUUUCACA
[2000-2018](19/19)


 


345
GUGAUGUGAAAUGCUCAUA
848
UAUGAGCAUUUCACAUCAC
[1996-2014](19/19)


 


346
AGGUCCUACUGUGAUGUGA
849
UCACAUCACAGUAGGAGCU
[1986-2004](19/19)


 


347
UCCUAGUGAAUACUCCCUG
850
CAGGGAGUAUUCACUAGGA
[1844-1862](19/19)


 


348
UUCUCCUAGUGAAUACUCC
851
GGAGUAUUCACUAGGAGAA
[1841-1859](19/19)


 


349
CAUGCUACGUGAUGAAGAU
852
AUCUUCAUCACGUAGCAUG
[1811-1829](19/19)


 


350
GCAUGCUACGUGAUGAAGA
853
UCUUCAUCACGUAGCAUGC
[1810-1828](19/19)


 


351
AGCACCUUAUAUCUCGAAG
854
CUUCGAGAUAUAAGGUGCU
[1785-1803](19/19)


 


352
AGAAAAAGGAGAAAAUGAC
855
GUCAUUUUCUCCUUUUUCU
[1736-1754](19/19)


 


353
UGCAGAAAAAGAAAACUGG
856
CCAGUUUUCUUUUUCUGCA
[1653-1671](19/19)


 


354
UCAUUAACCUCCCUGUUGU
857
ACAACAGGGAGGUUAAUGA
[1531-1549](19/19)


 


355
AAGUGUCAAACAGAAUGGU
858
ACCAUUCUGUUUGACACUU
[1271-1289](19/19)


 


356
GAAAGAGUAUGAGCUGGAA
859
UUCCAGCUCAUACUCUUUC
[242-260](19/19)


 


357
AAAGCACAGCAGAAUUCAA
860
UUGAAUUCUGCUGUGCUUU
[1114-1132](19/19)


 


358
AUGUUUCUGAUCUAUCACU
861
AGUGAUAGAUCAGAAACAU
[1066-1084](19/19)


 


359
UGGGCCCAUUGAUGUUUCU
862
AGAAACAUCAAUGGGCCCA
[1055-1073](19/19)


 


360
CAGUCAACACAGAUUUUGG
863
CCAAAAUCUGUGUUGACUG
[940-958](19/19)


 


361
ACAGUCAACACAGAUUUUG
864
CAAAAUCUGUGUUGACUGU
[939-957](19/19)


 


362
CACAGUCAACACAGAUUUU
865
AAAAUCUGUGUUGACUGUG
[938-956](19/19)


 


363
ACAGAAGACCCCAACCAGU
866
ACUGGUUGGGGUCUUCUGU
[891-909](19/19)


 


364
AGGAUUCCUUCAGCAGCAU
867
AUGCUGCUGAAGGAAUCCU
[865-883](19/19)


 


365
AAUGGAAAAAGAAGUAGGU
868
ACCUACUUCUUUUUCCAUU
[815-833](19/19)


 


366
AUGACAAGCUGGUUGAGAC
869
GUCUCAACCAGCUUGUCAU
[724-742](19/19)


 


367
UUGAGCAAGUUUGGGAGGA
870
UCCUCCCAAACUUGCUCAA
[664-682](19/19)


 


368
CUGAAACUUCUGUUGCUCA
871
UGAGCAACAGAAGUUUCAG
[607-625](19/19)


 


369
UCACCUGAAACUUCUGUUG
872
CAACAGAAGUUUCAGGUGA
[603-621](19/19)


 


370
UUGACAUACUUUGGAGGCA
873
UGCCUCCAAAGUAUGUCAA
[172-190](19/19)


 


371
UGAUUGACAUACUUUGGAG
874
CUCCAAAGUAUGUCAAUCA
[169-187](19/19)


 


372
AGGUUUCUUCGGCUACGUU
875
AACGUAGCCGAAGAAACCU
[514-532](19/19)


 


373
AUGGAUUUGAUUGACAUAC
876
GUAUGUCAAUCAAAUCCAU
[162-180](19/19)


 


374
ACAUGGAUUUGAUUGACAU
877
AUGUCAAUCAAAUCCAUGU
[160-178](19/19)


 


375
UUCUCCCAAUUCAGCCAGC
878
GCUGGCUGAAUUGGGAGAA
[361-379](19/19)


 


376
AAGAGACAGGUGAAUUUCU
879
AGAAAUUCACCUGUCUCUU
[346-364](19/19)


 


377
GAUGAAGAGACAGGUGAAU
880
AUUCACCUGUCUCUUCAUC
[342-360](19/19)


 


378
AACUAGAUGAAGAGACAGG
881
CCUGUCUCUUCAUCUAGUU
[337-355](19/19)


 


379
UUGCAAAACUAACCACUAU
882
AUAGUGGUUAGUUUUGCAA
[2268-2286](19/19)


 


380
UGACAUCUGGCUAAAAAGA
883
UCUUUUUAGCCAGAUGUCA
[2244-2262](19/19)


 


381
GAGUGUCAGUAUGUUGAAU
884
AUUCAACAUACUGACACUC
[2089-2107](19/19)


 


382
AAGCAUUGGAGUGUCAGUA
885
UACUGACACUCCAAUGCUU
[2081-2099](19/19)


 


383
GCUCCUACUGUGAUGUGAA
886
UUCACAUCACAGUAGGAGC
[1987-2005](19/19)


 


384
GACCUUUUCUGAGCUAGUU
887
AACUAGCUCAGAAAAGGUC
[1946-1964](19/19)


 


385
UGACCUUUUCUGAGCUAGU
888
ACUAGCUCAGAAAAGGUCA
[1945-1963](19/19)


 


386
UCAUGAUGGACUUGGAGCU
889
AGCUCCAAGUCCAUCAUGA
[112-130](19/19)


 


387
CCUUAUAUCUCGAAGUUUU
890
AAAACUUCGAGAUAUAAGG
[1789-1807](19/19)


 


388
CUCAGCACCUUAUAUCUCG
891
CGAGAUAUAAGGUGCUGAG
[1782-1800](19/19)


 


389
GACAAGAACAACUCCAAAA
892
UUUUGGAGUUGUUCUUGUC
[286-304](19/19)


 


390
UCGGGAUAUACGUAGGAGG
893
CCUCCUACGUAUAUCCCGA
[1607-1625](19/19)


 


391
UAAUUCGGGAUAUACGUAG
894
CUACGUAUAUCCCGAAUUA
[1603-1621](19/19)


 


392
GAAAUGAUGUCCAAAGAGC
895
GCUCUUUGGACAUCAUUUC
[1560-1578](19/19)


 


393
AACGAAAUGAUGUCCAAAG
896
CUUUGGACAUCAUUUCGUU
[1557-1575](19/19)


 


394
GAAAAAAUCAUUAACCUCC
897
GGAGGUUAAUGAUUUUUUC
[1524-1542](19/19)


 


395
AGCUCUCCAUAUCCCAUUC
898
GAAUGGGAUAUGGAGAGCU
[1499-1517](19/19)


 


396
GCAAAAGCUCUCCAUAUCC
899
GGAUAUGGAGAGCUUUUGC
[1494-1512](19/19)


 


397
AAGAGAUGAACUUAGGGCA
900
UGCCCUAAGUUCAUCUCUU
[1478-1496](19/19)


 


398
CUGGAAAAACAGAAAAAAC
901
GUUUUUUCUGUUUUUCCAG
[255-273](19/19)


 


399
GAGCUGGAAAAACAGAAAA
902
UUUUCUGUUUUUCCAGCUC
[252-270](19/19)


 


400
CCCCUGGAAGUGUCAAACA
903
UGUUUGACACUUCCAGGGG
[1264-1282](19/19)


 


401
AUUCUGAAGUGGAAGAGCU
904
AGCUCUUCCACUUCAGAAU
[1237-1255](19/19)


 


402
CAUCACCAGAACACUCAGU
905
ACUGAGUGUUCUGGUGAUG
[1177-1195](19/19)


 


403
CUGAUCUAUCACUUUGCAA
906
UUGCAAAGUGAUAGAUCAG
[1072-1090](19/19)


 


404
UCUGAUCUAUCACUUUGCA
907
UGCAAAGUGAUAGAUCAGA
[1071-1089](19/19)


 


405
UUCUGAUCUAUCACUUUGC
908
GCAAAGUGAUAGAUCAGAA
[1070-1088](19/19)


 


406
GAGCCCAGUAUCAGCAACA
909
UGUUGCUGAUACUGGGCUC
[987-1005](19/19)


 


407
GAAGUAGGUAACUGUAGUC
910
GACUACAGUUACCUACUUC
[825-843](19/19)


 


408
AGAAGUAGGUAACUGUAGU
911
ACUACAGUUACCUACUUCU
[824-842](19/19)


 


409
AAAAGAAGUAGGUAACUGU
912
ACAGUUACCUACUUCUUUU
[821-839](19/19)


 


410
GCAACAGGACAUUGAGCAA
913
UUGCUCAAUGUCCUGUUGC
[653-671](19/19)


 


411
CACUUGUUCCUGAUAUUCC
914
GGAAUAUCAGGAACAAGUG
[538-556](19/19)


 


412
GCAGGACAUGGAUUUGAUU
915
AAUCAAAUCCAUGUCCUGC
[155-173](19/19)


 


413
GUUUCUGUGUAAGUGUAAA
916
UUUACACUUACACAGAAAC
[2159-2177](19/19)


 


414
GCAUUGGAGUGUCAGUAUG
917
CAUACUGACACUCCAAUGC
[2083-2101](19/19)


 


415
UGUGAUGUGAAAUGCUCAU
918
AUGAGCAUUUCACAUCACA
[1995-2013](19/19)


 


416
ACUAGAUUUAGGAGGAUUU
919
AAAUCCUCCUAAAUCUAGU
[1927-1945](19/19)


 


417
CUCGAAGUUUUCAGCAUGC
920
GCAUGCUGAAAACUUCGAG
[1797-1815](19/19)


 


418
AUCUCGAAGUUUUCAGCAU
921
AUGCUGAAAACUUCGAGAU
[1795-1813](19/19)


 


419
GCAGAAAAAGAAAACUGGA
922
UCCAGUUUUCUUUUUCUGC
[1654-1672](19/19)


 


420
GGAUAUACGUAGGAGGGGU
923
ACCCCUCCUACGUAUAUCC
[1610-1628](19/19)


 


421
AAGACAAGAACAACUCCAA
924
UUGGAGUUGUUCUUGUCUU
[284-302](19/19)


 


422
AAAGAGCAGUUCAAUGAAG
925
CUUCAUUGAACUGCUCUUU
[1572-1590](19/19)


 


423
UCAACGAAAUGAUGUCCAA
926
UUGGACAUCAUUUCGUUGA
[1555-1573](19/19)


 


424
UAACCUCCCUGUUGUUGAC
927
GUCAACAACAGGGAGGUUA
[1535-1553](19/19)


 


425
CAAAAGCUCUCCAUAUCCC
928
GGGAUAUGGAGAGCUUUUG
[1495-1513](19/19)


 


426
AGAUGAACUUAGGGCAAAA
929
UUUUGCCCUAAGUUCAUCU
[1481-1499](19/19)


 


427
AGAGAUGAACUUAGGGCAA
930
UUGCCCUAAGUUCAUCUCU
[1479-1497](19/19)


 


428
AGCUGGAAAAACAGAAAAA
931
UUUUUCUGUUUUUCCAGCU
[253-271](19/19)


 


429
UCCUAAAACACCAGUACAU
932
AUGUACUGGUGUUUUAGGA
[1289-1307](19/19)


 


430
UGAUGUUUCUGAUCUAUCA
933
UGAUAGAUCAGAAACAUCA
[1064-1082](19/19)


 


431
CCAUUGAUGUUUCUGAUCU
934
AGAUCAGAAACAUCAAUGG
[1060-1078](19/19)


 


432
AGUAUGUUGAAUCAGUAGU
935
ACUACUGAUUCAACAUACU
[2096-2114](19/19)


 


433
UUCUGAGCUAGUUUUUUUG
936
CAAAAAAACUAGCUCAGAA
[1952-1970](19/19)


 


434
UCGAAGUUUUCAGCAUGCU
937
AGCAUGCUGAAAACUUCGA
[1798-1816](19/19)


 


435
AUUCGGGAUAUACGUAGGA
938
UCCUACGUAUAUCCCGAAU
[1605-1623](19/19)


 


436
UGUCCAAAGAGCAGUUCAA
939
UUGAACUGCUCUUUGGACA
[1567-1585](19/19)


 


437
UUCUGGGGAUAUGGUACAA
940
UUGUACCAUAUCCCCAGAA
[1310-1328](19/19)


 


438
CUUCUGGGGAUAUGGUACA
941
UGUACCAUAUCCCCAGAAG
[1309-1327](19/19)


 


439
UGUUUCUGAUCUAUCACUU
942
AAGUGAUAGAUCAGAAACA
[1067-1085](19/19)


 


440
ACCUGCUACUUUAAGCCAU
943
AUGGCUUAAAGUAGCAGGU
[1016-1034](19/19)


 


441
GUUUCAGUCACUUGUUCCU
944
AGGAACAAGUGACUGAAAC
[530-548](19/19)


 


442
CACAUCCAGUCAGAAACCA
945
UGGUUUCUGACUGGAUGUG
[384-402](19/19)


 


443
CUGGUACUAAUAAAGGAUU
946
AAUCCUUUAUUAGUACCAG
[2375-2393](19/19)


 


444
AGUGUCAGUAUGUUGAAUC
947
GAUUCAACAUACUGACACU
[2090-2108](19/19)


 


445
AAACUAGAUUUAGGAGGAU
948
AUCCUCCUAAAUCUAGUUU
[1925-1943](19/19)


 


446
AAAACUAGAUUUAGGAGGA
949
UCCUCCUAAAUCUAGUUUU
[1924-1942](19/19)


 


447
AGCUCAACUUGCAUUAAUU
950
AAUUAAUGCAAGUUGAGCU
[1589-1607](19/19)


 


448
AAGAGCAGUUCAAUGAAGC
951
GCUUCAUUGAACUGCUCUU
[1573-1591](19/19)


 


449
UGAUGUCCAAAGAGCAGUU
952
AACUGCUCUUUGGACAUCA
[1564-1582](19/19)


 


450
GAAAACCCCAUUCACAAAA
953
UUUUGUGAAUGGGGUUUUC
[1424-1442](19/19)


 


451
UGAGCUGGAAAAACAGAAA
954
UUUCUGUUUUUCCAGCUCA
[251-269](19/19)


 


452
UUGAUGUUUCUGAUCUAUC
955
GAUAGAUCAGAAACAUCAA
[1063-1081](19/19)


 


453
UUGCCCACAUUCCCAAAUC
956
GAUUUGGGAAUGUGGGCAA
[427-445](19/19)


 


454
GCACAUCCAGUCAGAAACC
957
GGUUUCUGACUGGAUGUGC
[383-401](19/19)


 


455
GCUGGUACUAAUAAAGGAU
958
AUCCUUUAUUAGUACCAGC
[2374-2392](19/19)


 


456
GGAGUGUCAGUAUGUUGAA
959
UUCAACAUACUGACACUCC
[2088-2106](19/19)


 


457
CCUUUUCUGAGCUAGUUUU
960
AAAACUAGCUCAGAAAAGG
[1948-1966](19/19)


 


458
CUCAAAGAAAAAGGAGAAA
961
UUUCUCCUUUUUCUUUGAG
[1731-1749](19/19)


 


459
AGACAAGAACAACUCCAAA
962
UUUGGAGUUGUUCUUGUCU
[285-303](19/19)


 


460
AUGAUGUCCAAAGAGCAGU
963
ACUGCUCUUUGGACAUCAU
[1563-1581](19/19)


 


461
AUGAGCUGGAAAAACAGAA
964
UUCUGUUUUUCCAGCUCAU
[250-268](19/19)


 


462
GUCCUAAAACACCAGUACA
965
UGUACUGGUGUUUUAGGAC
[1288-1306](19/19)


 


463
AUCAGCAACAGCAUGCCCU
966
AGGGCAUGCUGUUGCUGAU
[996-1014](19/19)


 


464
AAGCCAAACUGACAGAAGU
967
ACUUCUGUCAGUUUGGCUU
[763-781](19/19)


 


465
CGUUUGUAGAUGACAAUGA
968
UCAUUGUCAUCUACAAACG
[496-514](19/19)


 


466
GUUGCCCACAUUCCCAAAU
969
AUUUGGGAAUGUGGGCAAC
[426-444](19/19)


 


467
GCAAACAAGAGAUGGCAAU
970
AUUGCCAUCUCUUGUUUGC
[1865-1883](19/19)


 


468
AUUGCUCAAAGAAAAAGGA
971
UCCUUUUUCUUUGAGCAAU
[1727-1745](19/19)


 


469
AAACCCCAUUCACAAAAGA
972
UCUUUUGUGAAUGGGGUUU
[1426-1444](19/19)


 


470
AAGUGGAAGAGCUAGAUAG
973
CUAUCUAGCUCUUCCACUU
[1243-1261](19/19)


 


471
UCACUAAACACAAGUCCCA
974
UGGGACUUGUGUUUAGUGA
[1152-1170](19/19)


 


472
CGGCAUUUCACUAAACACA
975
UGUGUUUAGUGAAAUGCCG
[1145-1163](19/19)


 


473
UUUAAGCCAUUCACUCUCU
976
AGAGAGUGAAUGGCUUAAA
[1025-1043](19/19)


 


474
CACCUGCUACUUUAAGCCA
977
UGGCUUAAAGUAGCAGGUG
[1015-1033](19/19)


 


475
GGAGGAGCUAUUAUCCAUU
978
AAUGGAUAAUAGCUCCUCC
[677-695](19/19)


 


476
GGGAGGAGCUAUUAUCCAU
979
AUGGAUAAUAGCUCCUCCC
[676-694](19/19)


 


477
UCGCUCAGUUACAACUAGA
980
UCUAGUUGUAACUGAGCGA
[325-343](19/19)


 


478
UGAAGAUGGAAAACCUUAU
981
AUAAGGUUUUCCAUCUUCA
[1823-1841](19/19)


 


479
AAGCUCAACUUGCAUUAAU
982
AUUAAUGCAAGUUGAGCUU
[1588-1606](19/19)


 


480
AAAACCCCAUUCACAAAAG
983
CUUUUGUGAAUGGGGUUUU
[1425-1443](19/19)


 


481
GCUAUGGAGACACACUACU
984
AGUAGUGUGUCUCCAUAGC
[1207-1225](19/19)


 


482
GUAUUUGACUUCAGUCAGC
985
GCUGACUGAAGUCAAAUAC
[219-237](19/19)


 


483
CCUGCUACUUUAAGCCAUU
986
AAUGGCUUAAAGUAGCAGG
[1017-1035](19/19)


 


484
UGAGCCCAGUAUCAGCAAC
987
GUUGCUGAUACUGGGCUCA
[986-1004](19/19)


 


485
GAGAAGUAUUUGACUUCAG
988
CUGAAGUCAAAUACUUCUC
[214-232](19/19)


 


486
UGGAAAAAGAAGUAGGUAA
989
UUACCUACUUCUUUUUCCA
[817-835](19/19)


 


487
GAAGCCAAACUGACAGAAG
990
CUUCUGUCAGUUUGGCUUC
[762-780](19/19)


 


488
CAGAAGCCAAACUGACAGA
991
UCUGUCAGUUUGGCUUCUG
[760-778](19/19)


 


489
GAAAAUGACAAGCUGGUUG
992
CAACCAGCUUGUCAUUUUC
[720-738](19/19)


 


490
AAAAAGCAUUGGAGUGUCA
993
UGACACUCCAAUGCUUUUU
[2078-2096](19/19)


 


491
UAGGAGGAUUUGACCUUUU
994
AAAAGGUCAAAUCCUCCUA
[1935-1953](19/19)


 


492
AGAUGGAAAACCUUAUUCU
995
AGAAUAAGGUUUUCCAUCU
[1826-1844](19/19)


 


493
GAAGCUCAACUUGCAUUAA
996
UUAAUGCAAGUUGAGCUUC
[1587-1605](19/19)


 


494
GCUCUCCAUAUCCCAUUCC
997
GGAAUGGGAUAUGGAGAGC
[1500-1518](19/19)


 


495
GGAAAACCCCAUUCACAAA
998
UUUGUGAAUGGGGUUUUCC
[1423-1441](19/19)


 


496
AGAGCACUCACGUGCAUGA
999
UCAUGCACGUGAGUGCUCU
[1351-1369](19/19)


 


497
ACCCUUGUCACCAUCUCAG
1000
CUGAGAUGGUGACAAGGGU
[1328-1346](19/19)


 


498
CUACUUGGCCUCAGUGAUU
1001
AAUCACUGAGGCCAAGUAG
[1221-1239](19/19)


 


499
GUGAACUCAUUAAAUUCAG
1002
CUGAAUUUAAUGAGUUCAC
[915-933](19/19)


 


500
GGAAAAAGAAGUAGGUAAC
1003
GUUACCUACUUCUUUUUCC
[818-836](19/19)


 


501
CCCUGUCGAAAAAAUCAUU
1004
AAUGAUUUUUUCGACAGGG


 


502
UCCCUGUCGAAAAAAUCAU
1005
AUGAUUUUUUCGACAGGGA


 


503
CCUGUCGAAAAAAUCAUUA
1006
UAAUGAUUUUUUCGACAGG
















TABLE B







21-mers siRNAs specific to Nrf2 gene (SEQ ID NOS: 1007-2006)











SEQ ID NO.
Sense siRNA
SEQ ID NO.
Antisense siRNA
Human 20149575














1007
UAGGAGGGGUAAGAAUAAAGU
1507
ACUUUAUUCUUACCCCUCCUA
[1619-1639](21/21)


 


1008
AGGGGUAAGAAUAAAGUGGCU
1508
AGCCACUUUAUUCUUACCCCU
[1623-1643](21/21)


 


1009
AAGAAGCCAGAUGUUAAGAAA
1509
UUUCUUAACAUCUGGCUUCUU
[1905-1925](21/21)


 


1010
AAAGGAGAAAAUGACAAAAGC
1510
GCUUUUGUCAUUUUCUCCUUU
[1740-1760](21/21)


 


1011
GAGGGGUAAGAAUAAAGUGGC
1511
GCCACUUUAUUCUUACCCCUC
[1622-1642](21/21)


 


1012
GUAGGAGGGGUAAGAAUAAAG
1512
CUUUAUUCUUACCCCUCCUAC
[1618-1638](21/21)


 


1013
AUAAAGUGGCUGCUCAGAAUU
1513
AAUUCUGAGCAGCCACUUUAU
[1633-1653](21/21)


 


1014
AGGAGGGGUAAGAAUAAAGUG
1514
CACUUUAUUCUUACCCCUCCU
[1620-1640](21/21)


 


1015
GAGAAAGAAUUGCCUGUAAGU
1515
ACUUACAGGCAAUUCUUUCUC
[1392-1412](21/21)


 


1016
UCACUCUCUGAACUUCUAAAU
1516
AUUUAGAAGUUCAGAGAGUGA
[1035-1055](21/21)


 


1017
GACAUUCCCGUUUGUAGAUGA
1517
UCAUCUACAAACGGGAAUGUC
[488-508](21/21)


 


1018
AGAAGCCAGAUGUUAAGAAAA
1518
UUUUCUUAACAUCUGGCUUCU
[1906-1926](21/21)


 


1019
CUCAGAAUUGCAGAAAAAGAA
1519
UUCUUUUUCUGCAAUUCUGAG
[1645-1665](21/21)


 


1020
AAGAAUAAAGUGGCUGCUCAG
1520
CUGAGCAGCCACUUUAUUCUU
[1629-1649](21/21)


 


1021
GGGUAAGAAUAAAGUGGCUGC
1521
GCAGCCACUUUAUUCUUACCC
[1625-1645](21/21)


 


1022
UGAAAAGGAAAGACAAGAACA
1522
UGUUCUUGUCUUUCCUUUUCA
[275-295](21/21)


 


1023
GCCCUCACCUGCUACUUUAAG
1523
CUUAAAGUAGCAGGUGAGGGC
[1010-1030](21/21)


 


1024
GUAGAUGACAAUGAGGUUUCU
1524
AGAAACCUCAUUGUCAUCUAC
[501-521](21/21)


 


1025
GUAAGAAGCCAGAUGUUAAGA
1525
UCUUAACAUCUGGCUUCUUAC
[1903-1923](21/21)


 


1026
AAGUAAGAAGCCAGAUGUUAA
1526
UUAACAUCUGGCUUCUUACUU
[1901-1921](21/21)


 


1027
GGGGUAAGAAUAAAGUGGCUG
1527
CAGCCACUUUAUUCUUACCCC
[1624-1644](21/21)


 


1028
AGAGAAAGAAUUGCCUGUAAG
1528
CUUACAGGCAAUUCUUUCUCU
[1391-1411](21/21)


 


1029
CUCUCUGAACUUCUAAAUGGG
1529
CCCAUUUAGAAGUUCAGAGAG
[1038-1058](21/21)


 


1030
UUACUCAUCUAUACCCUCAAU
1530
AUUGAGGGUAUAGAUGAGUAA
[797-817](21/21)


 


1031
CCAGUCUUCAUUGCUACUAAU
1531
AUUAGUAGCAAUGAAGACUGG
[573-593](21/21)


 


1032
UAGAUGACAAUGAGGUUUCUU
1532
AAGAAACCUCAUUGUCAUCUA
[502-522](21/21)


 


1033
ACAUUCCCGUUUGUAGAUGAC
1533
GUCAUCUACAAACGGGAAUGU
[489-509](21/21)


 


1034
GCAAAACUAACCACUAUGUAC
1534
GUACAUAGUGGUUAGUUUUGC
[2270-2290](21/21)


 


1035
AAAAGUAAGAAGCCAGAUGUU
1535
AACAUCUGGCUUCUUACUUUU
[1899-1919](21/21)


 


1036
CCCAAAAGUAAGAAGCCAGAU
1536
AUCUGGCUUCUUACUUUUGGG
[1896-1916](21/21)


 


1037
UUGCUCAAAGAAAAAGGAGAA
1537
UUCUCCUUUUUCUUUGAGCAA
[1728-1748](21/21)


 


1038
GAACUAGAGCAAGAUUUAGAU
1538
AUCUAAAUCUUGCUCUAGUUC
[1683-1703](21/21)


 


1039
GCUCAGAAUUGCAGAAAAAGA
1539
UCUUUUUCUGCAAUUCUGAGC
[1644-1664](21/21)


 


1040
UUGAAAAGGAAAGACAAGAAC
1540
GUUCUUGUCUUUCCUUUUCAA
[274-294](21/21)


 


1041
AGAACACACCAGAGAAAGAAU
1541
AUUCUUUCUCUGGUGUGUUCU
[1381-1401](21/21)


 


1042
AAAACACCAGUACAUUCUUCU
1542
AGAAGAAUGUACUGGUGUUUU
[1293-1313](21/21)


 


1043
GUCAAACAGAAUGGUCCUAAA
1543
UUUAGGACCAUUCUGUUUGAC
[1275-1295](21/21)


 


1044
AACCAAAACCACCCUGAAAGC
1544
GCUUUCAGGGUGGUUUUGGUU
[1098-1118](21/21)


 


1045
UUCAACCAAAACCACCCUGAA
1545
UUCAGGGUGGUUUUGGUUGAA
[1095-1115](21/21)


 


1046
CACUCUCUGAACUUCUAAAUG
1546
CAUUUAGAAGUUCAGAGAGUG
[1036-1056](21/21)


 


1047
GAGUAAGUCGAGAAGUAUUUG
1547
CAAAUACUUCUCGACUUACUC
[205-225](21/21)


 


1048
GACAAUGAGGUUUCUUCGGCU
1548
AGCCGAAGAAACCUCAUUGUC
[507-527](21/21)


 


1049
UCAGAUGCUUUGUACUUUGAU
1549
AUCAAAGUACAAAGCAUCUGA
[444-464](21/21)


 


1050
GACAUCUGGCUAAAAAGAAAU
1550
AUUUCUUUUUAGCCAGAUGUC
[2245-2265](21/21)


 


1051
GAAGCCAGAUGUUAAGAAAAA
1551
UUUUUCUUAACAUCUGGCUUC
[1907-1927](21/21)


 


1052
CAAAAGUAAGAAGCCAGAUGU
1552
ACAUCUGGCUUCUUACUUUUG
[1898-1918](21/21)


 


1053
AAAACCUUAUUCUCCUAGUGA
1553
UCACUAGGAGAAUAAGGUUUU
[1832-1852](21/21)


 


1054
ACGUAGGAGGGGUAAGAAUAA
1554
UUAUUCUUACCCCUCCUACGU
[1616-1636](21/21)


 


1055
AAGGAAAGACAAGAACAACUC
1555
GAGUUGUUCUUGUCUUUCCUU
[279-299](21/21)


 


1056
CACAAAAGACAAACAUUCAAG
1556
CUUGAAUGUUUGUCUUUUGUG
[1436-1456](21/21)


 


1057
CCCAUUCACAAAAGACAAACA
1557
UGUUUGUCUUUUGUGAAUGGG
[1430-1450](21/21)


 


1058
AACCCCAUUCACAAAAGACAA
1558
UUGUCUUUUGUGAAUGGGGUU
[1427-1447](21/21)


 


1059
ACACCAGAGAAAGAAUUGCCU
1559
AGGCAAUUCUUUCUCUGGUGU
[1386-1406](21/21)


 


1060
AACACACCAGAGAAAGAAUUG
1560
CAAUUCUUUCUCUGGUGUGUU
[1383-1403](21/21)


 


1061
UCAAACAGAAUGGUCCUAAAA
1561
UUUUAGGACCAUUCUGUUUGA
[1276-1296](21/21)


 


1062
UCAGCGACGGAAAGAGUAUGA
1562
UCAUACUCUUUCCGUCGCUGA
[233-253](21/21)


 


1063
GCACAGCAGAAUUCAAUGAUU
1563
AAUCAUUGAAUUCUGCUGUGC
[1117-1137](21/21)


 


1064
GCUUUCAACCAAAACCACCCU
1564
AGGGUGGUUUUGGUUGAAAGC
[1092-1112](21/21)


 


1065
GCCACAGUCAACACAGAUUUU
1565
AAAAUCUGUGUUGACUGUGGC
[936-956](21/21)


 


1066
AACUCAUUAAAUUCAGAUGCC
1566
GGCAUCUGAAUUUAAUGAGUU
[918-938](21/21)


 


1067
GUUGACAGUGAACUCAUUAAA
1567
UUUAAUGAGUUCACUGUCAAC
[908-928](21/21)


 


1068
GCUAUUAUCCAUUCCUGAGUU
1568
AACUCAGGAAUGGAUAAUAGC
[683-703](21/21)


 


1069
AAGUUUGGGAGGAGCUAUUAU
1569
AUAAUAGCUCCUCCCAAACUU
[670-690](21/21)


 


1070
CAAGUUUGGGAGGAGCUAUUA
1570
UAAUAGCUCCUCCCAAACUUG
[669-689](21/21)


 


1071
GUCUUCAUUGCUACUAAUCAG
1571
CUGAUUAGUAGCAAUGAAGAC
[576-596](21/21)


 


1072
GAUGACAAUGAGGUUUCUUCG
1572
CGAAGAAACCUCAUUGUCAUC
[504-524](21/21)


 


1073
AUCAGAUGCUUUGUACUUUGA
1573
UCAAAGUACAAAGCAUCUGAU
[443-463](21/21)


 


1074
GCCCACAUUCCCAAAUCAGAU
1574
AUCUGAUUUGGGAAUGUGGGC
[429-449](21/21)


 


1075
GUGAUGUGAAAUGCUCAUACU
1575
AGUAUGAGCAUUUCACAUCAC
[1996-2016](21/21)


 


1076
AAGCUCCUACUGUGAUGUGAA
1576
UUCACAUCACAGUAGGAGCUU
[1985-2005](21/21)


 


1077
CUACGUGAUGAAGAUGGAAAA
1577
UUUUCCAUCUUCAUCACGUAG
[1815-1835](21/21)


 


1078
AUGCUACGUGAUGAAGAUGGA
1578
UCCAUCUUCAUCACGUAGCAU
[1812-1832](21/21)


 


1079
UGAAAAAACAACUCAGCACCU
1579
AGGUGCUGAGUUGUUUUUUCA
[1771-1791](21/21)


 


1080
UCACCUACUGAAAAAACAACU
1580
AGUUGUUUUUUCAGUAGGUGA
[1763-1783](21/21)


 


1081
GUAGAACUAGAGCAAGAUUUA
1581
UAAAUCUUGCUCUAGUUCUAC
[1680-1700](21/21)


 


1082
UCAACUUGCAUUAAUUCGGGA
1582
UCCCGAAUUAAUGCAAGUUGA
[1592-1612](21/21)


 


1083
AAAGGAAAGACAAGAACAACU
1583
AGUUGUUCUUGUCUUUCCUUU
[278-298](21/21)


 


1084
CCAUUCACAAAAGACAAACAU
1584
AUGUUUGUCUUUUGUGAAUGG
[1431-1451](21/21)


 


1085
CACACCAGAGAAAGAAUUGCC
1585
GGCAAUUCUUUCUCUGGUGUG
[1385-1405](21/21)


 


1086
AACAGAAUGGUCCUAAAACAC
1586
GUGUUUUAGGACCAUUCUGUU
[1279-1299](21/21)


 


1087
GGAAAGAGUAUGAGCUGGAAA
1587
UUUCCAGCUCAUACUCUUUCC
[241-261](21/21)


 


1088
CAGUUGACAGUGAACUCAUUA
1588
UAAUGAGUUCACUGUCAACUG
[906-926](21/21)


 


1089
GGAGUAAGUCGAGAAGUAUUU
1589
AAAUACUUCUCGACUUACUCC
[204-224](21/21)


 


1090
UGGAGUAAGUCGAGAAGUAUU
1590
AAUACUUCUCGACUUACUCCA
[203-223](21/21)


 


1091
UUGGAGUAAGUCGAGAAGUAU
1591
AUACUUCUCGACUUACUCCAA
[202-222](21/21)


 


1092
CUAUUAUCCAUUCCUGAGUUA
1592
UAACUCAGGAAUGGAUAAUAG
[684-704](21/21)


 


1093
GCAACAGGACAUUGAGCAAGU
1593
ACUUGCUCAAUGUCCUGUUGC
[653-673](21/21)


 


1094
GUUGAUUUAGACGGUAUGCAA
1594
UUGCAUACCGUCUAAAUCAAC
[636-656](21/21)


 


1095
CAGUCUUCAUUGCUACUAAUC
1595
GAUUAGUAGCAAUGAAGACUG
[574-594](21/21)


 


1096
UCACAUCGAGAGCCCAGUCUU
1596
AAGACUGGGCUCUCGAUGUGA
[560-580](21/21)


 


1097
AGGACAUGGAUUUGAUUGACA
1597
UGUCAAUCAAAUCCAUGUCCU
[157-177](21/21)


 


1098
UGCCCACAUUCCCAAAUCAGA
1598
UCUGAUUUGGGAAUGUGGGCA
[428-448](21/21)


 


1099
UACUCCCAGGUUGCCCACAUU
1599
AAUGUGGGCAACCUGGGAGUA
[417-437](21/21)


 


1100
AAGAGCUGGUACUAAUAAAGG
1600
CCUUUAUUAGUACCAGCUCUU
[2370-2390](21/21)


 


1101
UAAGAGCUGGUACUAAUAAAG
1601
CUUUAUUAGUACCAGCUCUUA
[2369-2389](21/21)


 


1102
CUGUAUGGACAAAAAAUGGCA
1602
UGCCAUUUUUUGUCCAUACAG
[2304-2324](21/21)


 


1103
GGAUUUGACCUUUUCUGAGCU
1603
AGCUCAGAAAAGGUCAAAUCC
[1940-1960](21/21)


 


1104
AAACCUUAUUCUCCUAGUGAA
1604
UUCACUAGGAGAAUAAGGUUU
[1833-1853](21/21)


 


1105
AAAAAACAACUCAGCACCUUA
1605
UAAGGUGCUGAGUUGUUUUUU
[1773-1793](21/21)


 


1106
CUAGAGCAAGAUUUAGAUCAU
1606
AUGAUCUAAAUCUUGCUCUAG
[1686-1706](21/21)


 


1107
GCAGAAAAAGAAAACUGGAAA
1607
UUUCCAGUUUUCUUUUUCUGC
[1654-1674](21/21)


 


1108
AACUUGCAUUAAUUCGGGAUA
1608
UAUCCCGAAUUAAUGCAAGUU
[1594-1614](21/21)


 


1109
CAACUUGCAUUAAUUCGGGAU
1609
AUCCCGAAUUAAUGCAAGUUG
[1593-1613](21/21)


 


1110
CCAUAUCCCAUUCCCUGUAGA
1610
UCUACAGGGAAUGGGAUAUGG
[1505-1525](21/21)


 


1111
ACAAAAGACAAACAUUCAAGC
1611
GCUUGAAUGUUUGUCUUUUGU
[1437-1457](21/21)


 


1112
ACCCCAUUCACAAAAGACAAA
1612
UUUGUCUUUUGUGAAUGGGGU
[1428-1448](21/21)


 


1113
AAGAAUUGCCUGUAAGUCCUG
1613
CAGGACUUACAGGCAAUUCUU
[1396-1416](21/21)


 


1114
AAAGAAUUGCCUGUAAGUCCU
1614
AGGACUUACAGGCAAUUCUUU
[1395-1415](21/21)


 


1115
GAACACACCAGAGAAAGAAUU
1615
AAUUCUUUCUCUGGUGUGUUC
[1382-1402](21/21)


 


1116
AGAGUAUGAGCUGGAAAAACA
1616
UGUUUUUCCAGCUCAUACUCU
[245-265](21/21)


 


1117
GUAGUCCACAUUUUCUUAAUG
1617
CAUUAAGAAAAUGUGGACUAC
[838-858](21/21)


 


1118
UACUCAUCUAUACCCUCAAUG
1618
CAUUGAGGGUAUAGAUGAGUA
[798-818](21/21)


 


1119
CAGGACAUUGAGCAAGUUUGG
1619
CCAAACUUGCUCAAUGUCCUG
[657-677](21/21)


 


1120
UUCGGCUACGUUUCAGUCACU
1620
AGUGACUGAAACGUAGCCGAA
[521-541](21/21)


 


1121
ACAAUGAGGUUUCUUCGGCUA
1621
UAGCCGAAGAAACCUCAUUGU
[508-528](21/21)


 


1122
GGACAUGGAUUUGAUUGACAU
1622
AUGUCAAUCAAAUCCAUGUCC
[158-178](21/21)


 


1123
UACUGUAUGGACAAAAAAUGG
1623
CCAUUUUUUGUCCAUACAGUA
[2302-2322](21/21)


 


1124
AACAAUUUCUUAGGACACCAU
1624
AUGGUGUCCUAAGAAAUUGUU
[2130-2150](21/21)


 


1125
CUGUAAACAAUUUCUUAGGAC
1625
GUCCUAAGAAAUUGUUUACAG
[2125-2145](21/21)


 


1126
GAAGAUGGAAAACCUUAUUCU
1626
AGAAUAAGGUUUUCCAUCUUC
[1824-1844](21/21)


 


1127
UACGUGAUGAAGAUGGAAAAC
1627
GUUUUCCAUCUUCAUCACGUA
[1816-1836](21/21)


 


1128
AGCACCUUAUAUCUCGAAGUU
1628
AACUUCGAGAUAUAAGGUGCU
[1785-1805](21/21)


 


1129
AAAACAACUCAGCACCUUAUA
1629
UAUAAGGUGCUGAGUUGUUUU
[1775-1795](21/21)


 


1130
GAGCAAGAUUUAGAUCAUUUG
1630
CAAAUGAUCUAAAUCUUGCUC
[1689-1709](21/21)


 


1131
AGAACUAGAGCAAGAUUUAGA
1631
UCUAAAUCUUGCUCUAGUUCU
[1682-1702](21/21)


 


1132
UUGCAGAAAAAGAAAACUGGA
1632
UCCAGUUUUCUUUUUCUGCAA
[1652-1672](21/21)


 


1133
UCCAUAUCCCAUUCCCUGUAG
1633
CUACAGGGAAUGGGAUAUGGA
[1504-1524](21/21)


 


1134
CCCCAUUCACAAAAGACAAAC
1634
GUUUGUCUUUUGUGAAUGGGG
[1429-1449](21/21)


 


1135
GAGAACACACCAGAGAAAGAA
1635
UUCUUUCUCUGGUGUGUUCUC
[1380-1400](21/21)


 


1136
CAGUACAUUCUUCUGGGGAUA
1636
UAUCCCCAGAAGAAUGUACUG
[1300-1320](21/21)


 


1137
ACGGAAAGAGUAUGAGCUGGA
1637
UCCAGCUCAUACUCUUUCCGU
[239-259](21/21)


 


1138
CUGACUCCGGCAUUUCACUAA
1638
UUAGUGAAAUGCCGGAGUCAG
[1138-1158](21/21)


 


1139
AGCACAGCAGAAUUCAAUGAU
1639
AUCAUUGAAUUCUGCUGUGCU
[1116-1136](21/21)


 


1140
UAAAUUCAGAUGCCACAGUCA
1640
UGACUGUGGCAUCUGAAUUUA
[925-945](21/21)


 


1141
CUCAUUAAAUUCAGAUGCCAC
1641
GUGGCAUCUGAAUUUAAUGAG
[920-940](21/21)


 


1142
ACUGUAGUCCACAUUUUCUUA
1642
UAAGAAAAUGUGGACUACAGU
[835-855](21/21)


 


1143
UCAAUGGAAAAAGAAGUAGGU
1643
ACCUACUUCUUUUUCCAUUGA
[813-833](21/21)


 


1144
GAGCUAUUAUCCAUUCCUGAG
1644
CUCAGGAAUGGAUAAUAGCUC
[681-701](21/21)


 


1145
AACAGGACAUUGAGCAAGUUU
1645
AAACUUGCUCAAUGUCCUGUU
[655-675](21/21)


 


1146
UUAGACGGUAUGCAACAGGAC
1646
GUCCUGUUGCAUACCGUCUAA
[642-662](21/21)


 


1147
UUUAGACGGUAUGCAACAGGA
1647
UCCUGUUGCAUACCGUCUAAA
[641-661](21/21)


 


1148
UGAGGUUUCUUCGGCUACGUU
1648
AACGUAGCCGAAGAAACCUCA
[512-532](21/21)


 


1149
AUGAGGUUUCUUCGGCUACGU
1649
ACGUAGCCGAAGAAACCUCAU
[511-531](21/21)


 


1150
UGCUUUGUACUUUGAUGACUG
1650
CAGUCAUCAAAGUACAAAGCA
[449-469](21/21)


 


1151
AGAUGCUUUGUACUUUGAUGA
1651
UCAUCAAAGUACAAAGCAUCU
[446-466](21/21)


 


1152
CAGCAGGACAUGGAUUUGAUU
1652
AAUCAAAUCCAUGUCCUGCUG
[153-173](21/21)


 


1153
UUACAACUAGAUGAAGAGACA
1653
UGUCUCUUCAUCUAGUUGUAA
[333-353](21/21)


 


1154
AGAGCUGGUACUAAUAAAGGA
1654
UCCUUUAUUAGUACCAGCUCU
[2371-2391](21/21)


 


1155
UGACAUCUGGCUAAAAAGAAA
1655
UUUCUUUUUAGCCAGAUGUCA
[2244-2264](21/21)


 


1156
CUAGUUUCUGUGUAAGUGUAA
1656
UUACACUUACACAGAAACUAG
[2156-2176](21/21)


 


1157
GGCUAGUUUCUGUGUAAGUGU
1657
ACACUUACACAGAAACUAGCC
[2154-2174](21/21)


 


1158
UGGGCUAGUUUCUGUGUAAGU
1658
ACUUACACAGAAACUAGCCCA
[2152-2172](21/21)


 


1159
AAACAAUUUCUUAGGACACCA
1659
UGGUGUCCUAAGAAAUUGUUU
[2129-2149](21/21)


 


1160
GAGUGUCAGUAUGUUGAAUCA
1660
UGAUUCAACAUACUGACACUC
[2089-2109](21/21)


 


1161
UACUAAAAGCUCCUACUGUGA
1661
UCACAGUAGGAGCUUUUAGUA
[1979-1999](21/21)


 


1162
UGCUACGUGAUGAAGAUGGAA
1662
UUCCAUCUUCAUCACGUAGCA
[1813-1833](21/21)


 


1163
GCACCUUAUAUCUCGAAGUUU
1663
AAACUUCGAGAUAUAAGGUGC
[1786-1806](21/21)


 


1164
CAGCACCUUAUAUCUCGAAGU
1664
ACUUCGAGAUAUAAGGUGCUG
[1784-1804](21/21)


 


1165
CACCUACUGAAAAAACAACUC
1665
GAGUUGUUUUUUCAGUAGGUG
[1764-1784](21/21)


 


1166
AACUAGAGCAAGAUUUAGAUC
1666
GAUCUAAAUCUUGCUCUAGUU
[1684-1704](21/21)


 


1167
UAGUAGAACUAGAGCAAGAUU
1667
AAUCUUGCUCUAGUUCUACUA
[1678-1698](21/21)


 


1168
GGGAUAUACGUAGGAGGGGUA
1668
UACCCCUCCUACGUAUAUCCC
[1609-1629](21/21)


 


1169
UGAGAACACACCAGAGAAAGA
1669
UCUUUCUCUGGUGUGUUCUCA
[1379-1399](21/21)


 


1170
AAAGAGUAUGAGCUGGAAAAA
1670
UUUUUCCAGCUCAUACUCUUU
[243-263](21/21)


 


1171
CGGAAAGAGUAUGAGCUGGAA
1671
UUCCAGCUCAUACUCUUUCCG
[240-260](21/21)


 


1172
UGCCCCUGGAAGUGUCAAACA
1672
UGUUUGACACUUCCAGGGGCA
[1262-1282](21/21)


 


1173
AUAGUGCCCCUGGAAGUGUCA
1673
UGACACUUCCAGGGGCACUAU
[1258-1278](21/21)


 


1174
AGCGACGGAAAGAGUAUGAGC
1674
GCUCAUACUCUUUCCGUCGCU
[235-255](21/21)


 


1175
CAGCGACGGAAAGAGUAUGAG
1675
CUCAUACUCUUUCCGUCGCUG
[234-254](21/21)


 


1176
GGGCCCAUUGAUGUUUCUGAU
1676
AUCAGAAACAUCAAUGGGCCC
[1056-1076](21/21)


 


1177
CUGUAGUCCACAUUUUCUUAA
1677
UUAAGAAAAUGUGGACUACAG
[836-856](21/21)


 


1178
CUCAAUGGAAAAAGAAGUAGG
1678
CCUACUUCUUUUUCCAUUGAG
[812-832](21/21)


 


1179
GAUCUUGGAGUAAGUCGAGAA
1679
UUCUCGACUUACUCCAAGAUC
[198-218](21/21)


 


1180
CUUUGGAGGCAAGAUAUAGAU
1680
AUCUAUAUCUUGCCUCCAAAG
[180-200](21/21)


 


1181
GACAUUGAGCAAGUUUGGGAG
1681
CUCCCAAACUUGCUCAAUGUC
[660-680](21/21)


 


1182
GGACAUUGAGCAAGUUUGGGA
1682
UCCCAAACUUGCUCAAUGUCC
[659-679](21/21)


 


1183
GUCACCUGAAACUUCUGUUGC
1683
GCAACAGAAGUUUCAGGUGAC
[602-622](21/21)


 


1184
GCCCAGUCUUCAUUGCUACUA
1684
UAGUAGCAAUGAAGACUGGGC
[571-591](21/21)


 


1185
UCGAGAGCCCAGUCUUCAUUG
1685
CAAUGAAGACUGGGCUCUCGA
[565-585](21/21)


 


1186
UCGGCUACGUUUCAGUCACUU
1686
AAGUGACUGAAACGUAGCCGA
[522-542](21/21)


 


1187
GAGGUUUCUUCGGCUACGUUU
1687
AAACGUAGCCGAAGAAACCUC
[513-533](21/21)


 


1188
UUGUACUUUGAUGACUGCAUG
1688
CAUGCAGUCAUCAAAGUACAA
[453-473](21/21)


 


1189
AUGCUUUGUACUUUGAUGACU
1689
AGUCAUCAAAGUACAAAGCAU
[448-468](21/21)


 


1190
GAUGCUUUGUACUUUGAUGAC
1690
GUCAUCAAAGUACAAAGCAUC
[447-467](21/21)


 


1191
CUAGAUGAAGAGACAGGUGAA
1691
UUCACCUGUCUCUUCAUCUAG
[339-359](21/21)


 


1192
GUAUGGACAAAAAAUGGCAUU
1692
AAUGCCAUUUUUUGUCCAUAC
[2306-2326](21/21)


 


1193
UGUAUGGACAAAAAAUGGCAU
1693
AUGCCAUUUUUUGUCCAUACA
[2305-2325](21/21)


 


1194
ACAAUUUCUUAGGACACCAUU
1694
AAUGGUGUCCUAAGAAAUUGU
[2131-2151](21/21)


 


1195
UGAUGUGAAAUGCUCAUACUU
1695
AAGUAUGAGCAUUUCACAUCA
[1997-2017](21/21)


 


1196
UCCUACUGUGAUGUGAAAUGC
1696
GCAUUUCACAUCACAGUAGGA
[1989-2009](21/21)


 


1197
UCCUAGUGAAUACUCCCUGCA
1697
UGCAGGGAGUAUUCACUAGGA
[1844-1864](21/21)


 


1198
ACACGGUCCACAGCUCAUCAU
1698
AUGAUGAGCUGUGGACCGUGU
[95-115](21/21)


 


1199
GAAGUGUCAAACAGAAUGGUC
1699
GACCAUUCUGUUUGACACUUC
[1270-1290](21/21)


 


1200
UGGAAGUGUCAAACAGAAUGG
1700
CCAUUCUGUUUGACACUUCCA
[1268-1288](21/21)


 


1201
GUGAUUCUGAAGUGGAAGAGC
1701
GCUCUUCCACUUCAGAAUCAC
[1234-1254](21/21)


 


1202
GAAAGCACAGCAGAAUUCAAU
1702
AUUGAAUUCUGCUGUGCUUUC
[1113-1133](21/21)


 


1203
CACAGUCAACACAGAUUUUGG
1703
CCAAAAUCUGUGUUGACUGUG
[938-958](21/21)


 


1204
CAGAAGCCAAACUGACAGAAG
1704
CUUCUGUCAGUUUGGCUUCUG
[760-780](21/21)


 


1205
ACCUGAAACUUCUGUUGCUCA
1705
UGAGCAACAGAAGUUUCAGGU
[605-625](21/21)


 


1206
UUGACAUACUUUGGAGGCAAG
1706
CUUGCCUCCAAAGUAUGUCAA
[172-192](21/21)


 


1207
UUGAUUGACAUACUUUGGAGG
1707
CCUCCAAAGUAUGUCAAUCAA
[168-188](21/21)


 


1208
GUCACUUGUUCCUGAUAUUCC
1708
GGAAUAUCAGGAACAAGUGAC
[536-556](21/21)


 


1209
GCUUUGUACUUUGAUGACUGC
1709
GCAGUCAUCAAAGUACAAAGC
[450-470](21/21)


 


1210
CAGGACAUGGAUUUGAUUGAC
1710
GUCAAUCAAAUCCAUGUCCUG
[156-176](21/21)


 


1211
CAGUUACAACUAGAUGAAGAG
1711
CUCUUCAUCUAGUUGUAACUG
[330-350](21/21)


 


1212
GAUAUGACAUCUGGCUAAAAA
1712
UUUUUAGCCAGAUGUCAUAUC
[2240-2260](21/21)


 


1213
CAGUAUGUUGAAUCAGUAGUU
1713
AACUACUGAUUCAACAUACUG
[2095-2115](21/21)


 


1214
AGCAUUGGAGUGUCAGUAUGU
1714
ACAUACUGACACUCCAAUGCU
[2082-2102](21/21)


 


1215
AAAGCAUUGGAGUGUCAGUAU
1715
AUACUGACACUCCAAUGCUUU
[2080-2100](21/21)


 


1216
CCUACUGUGAUGUGAAAUGCU
1716
AGCAUUUCACAUCACAGUAGG
[1990-2010](21/21)


 


1217
UGACCUUUUCUGAGCUAGUUU
1717
AAACUAGCUCAGAAAAGGUCA
[1945-1965](21/21)


 


1218
GAUUUGACCUUUUCUGAGCUA
1718
UAGCUCAGAAAAGGUCAAAUC
[1941-1961](21/21)


 


1219
CAGCAUGCUACGUGAUGAAGA
1719
UCUUCAUCACGUAGCAUGCUG
[1808-1828](21/21)


 


1220
CACCUUAUAUCUCGAAGUUUU
1720
AAAACUUCGAGAUAUAAGGUG
[1787-1807](21/21)


 


1221
UCAGCACCUUAUAUCUCGAAG
1721
CUUCGAGAUAUAAGGUGCUGA
[1783-1803](21/21)


 


1222
ACUCAGCACCUUAUAUCUCGA
1722
UCGAGAUAUAAGGUGCUGAGU
[1781-1801](21/21)


 


1223
AUAGUAGAACUAGAGCAAGAU
1723
AUCUUGCUCUAGUUCUACUAU
[1677-1697](21/21)


 


1224
GGAUAUACGUAGGAGGGGUAA
1724
UUACCCCUCCUACGUAUAUCC
[1610-1630](21/21)


 


1225
AAAGCUCUCCAUAUCCCAUUC
1725
GAAUGGGAUAUGGAGAGCUUU
[1497-1517](21/21)


 


1226
GCAAAAGCUCUCCAUAUCCCA
1726
UGGGAUAUGGAGAGCUUUUGC
[1494-1514](21/21)


 


1227
AGAGAUGAACUUAGGGCAAAA
1727
UUUUGCCCUAAGUUCAUCUCU
[1479-1499](21/21)


 


1228
CAAGAGAUGAACUUAGGGCAA
1728
UUGCCCUAAGUUCAUCUCUUG
[1477-1497](21/21)


 


1229
UCACAAGAGAUGAACUUAGGG
1729
CCCUAAGUUCAUCUCUUGUGA
[1474-1494](21/21)


 


1230
GGAAGUGUCAAACAGAAUGGU
1730
ACCAUUCUGUUUGACACUUCC
[1269-1289](21/21)


 


1231
CCUGGAAGUGUCAAACAGAAU
1731
AUUCUGUUUGACACUUCCAGG
[1266-1286](21/21)


 


1232
GAUUCUGAAGUGGAAGAGCUA
1732
UAGCUCUUCCACUUCAGAAUC
[1236-1256](21/21)


 


1233
GCGACGGAAAGAGUAUGAGCU
1733
AGCUCAUACUCUUUCCGUCGC
[236-256](21/21)


 


1234
UUCUGAUCUAUCACUUUGCAA
1734
UUGCAAAGUGAUAGAUCAGAA
[1070-1090](21/21)


 


1235
AUCUUGGAGUAAGUCGAGAAG
1735
CUUCUCGACUUACUCCAAGAU
[199-219](21/21)


 


1236
CAACAGGACAUUGAGCAAGUU
1736
AACUUGCUCAAUGUCCUGUUG
[654-674](21/21)


 


1237
UCACCUGAAACUUCUGUUGCU
1737
AGCAACAGAAGUUUCAGGUGA
[603-623](21/21)


 


1238
AUCGAGAGCCCAGUCUUCAUU
1738
AAUGAAGACUGGGCUCUCGAU
[564-584](21/21)


 


1239
UACGUUUCAGUCACUUGUUCC
1739
GGAACAAGUGACUGAAACGUA
[527-547](21/21)


 


1240
GCUACGUUUCAGUCACUUGUU
1740
AACAAGUGACUGAAACGUAGC
[525-545](21/21)


 


1241
GGCUACGUUUCAGUCACUUGU
1741
ACAAGUGACUGAAACGUAGCC
[524-544](21/21)


 


1242
CGGCUACGUUUCAGUCACUUG
1742
CAAGUGACUGAAACGUAGCCG
[523-543](21/21)


 


1243
UUUCUUCGGCUACGUUUCAGU
1743
ACUGAAACGUAGCCGAAGAAA
[517-537](21/21)


 


1244
GACAUGGAUUUGAUUGACAUA
1744
UAUGUCAAUCAAAUCCAUGUC
[159-179](21/21)


 


1245
AUAUGACAUCUGGCUAAAAAG
1745
CUUUUUAGCCAGAUGUCAUAU
[2241-2261](21/21)


 


1246
UUGGGCUAGUUUCUGUGUAAG
1746
CUUACACAGAAACUAGCCCAA
[2151-2171](21/21)


 


1247
GUCAGUAUGUUGAAUCAGUAG
1747
CUACUGAUUCAACAUACUGAC
[2093-2113](21/21)


 


1248
UGUCAGUAUGUUGAAUCAGUA
1748
UACUGAUUCAACAUACUGACA
[2092-2112](21/21)


 


1249
GUGUCAGUAUGUUGAAUCAGU
1749
ACUGAUUCAACAUACUGACAC
[2091-2111](21/21)


 


1250
UUGGAGUGUCAGUAUGUUGAA
1750
UUCAACAUACUGACACUCCAA
[2086-2106](21/21)


 


1251
UACUGUGAUGUGAAAUGCUCA
1751
UGAGCAUUUCACAUCACAGUA
[1992-2012](21/21)


 


1252
ACCUUUUCUGAGCUAGUUUUU
1752
AAAAACUAGCUCAGAAAAGGU
[1947-1967](21/21)


 


1253
UUUGACCUUUUCUGAGCUAGU
1753
ACUAGCUCAGAAAAGGUCAAA
[1943-1963](21/21)


 


1254
UGCAGCAAACAAGAGAUGGCA
1754
UGCCAUCUCUUGUUUGCUGCA
[1861-1881](21/21)


 


1255
CUCCUAGUGAAUACUCCCUGC
1755
GCAGGGAGUAUUCACUAGGAG
[1843-1863](21/21)


 


1256
GCAUGCUACGUGAUGAAGAUG
1756
CAUCUUCAUCACGUAGCAUGC
[1810-1830](21/21)


 


1257
ACCUUAUAUCUCGAAGUUUUC
1757
GAAAACUUCGAGAUAUAAGGU
[1788-1808](21/21)


 


1258
UUAAUUCGGGAUAUACGUAGG
1758
CCUACGUAUAUCCCGAAUUAA
[1602-1622](21/21)


 


1259
CAACGAAAUGAUGUCCAAAGA
1759
UCUUUGGACAUCAUUUCGUUG
[1556-1576](21/21)


 


1260
AACCUCCCUGUUGUUGACUUC
1760
GAAGUCAACAACAGGGAGGUU
[1536-1556](21/21)


 


1261
GAGAUGAACUUAGGGCAAAAG
1761
CUUUUGCCCUAAGUUCAUCUC
[1480-1500](21/21)


 


1262
ACAAGAGAUGAACUUAGGGCA
1762
UGCCCUAAGUUCAUCUCUUGU
[1476-1496](21/21)


 


1263
AAAGCACAGCAGAAUUCAAUG
1763
CAUUGAAUUCUGCUGUGCUUU
[1114-1134](21/21)


 


1264
GAUGUUUCUGAUCUAUCACUU
1764
AAGUGAUAGAUCAGAAACAUC
[1065-1085](21/21)


 


1265
ACAGUCAACACAGAUUUUGGU
1765
ACCAAAAUCUGUGUUGACUGU
[939-959](21/21)


 


1266
UGAGGAUUCCUUCAGCAGCAU
1766
AUGCUGCUGAAGGAAUCCUCA
[863-883](21/21)


 


1267
UGACAUACUUUGGAGGCAAGA
1767
UCUUGCCUCCAAAGUAUGUCA
[173-193](21/21)


 


1268
CAUGGAUUUGAUUGACAUACU
1768
AGUAUGUCAAUCAAAUCCAUG
[161-181](21/21)


 


1269
GCAGGACAUGGAUUUGAUUGA
1769
UCAAUCAAAUCCAUGUCCUGC
[155-175](21/21)


 


1270
AUGAAGAGACAGGUGAAUUUC
1770
GAAAUUCACCUGUCUCUUCAU
[343-363](21/21)


 


1271
AGAUGAAGAGACAGGUGAAUU
1771
AAUUCACCUGUCUCUUCAUCU
[341-361](21/21)


 


1272
UUGCAAAACUAACCACUAUGU
1772
ACAUAGUGGUUAGUUUUGCAA
[2268-2288](21/21)


 


1273
AUGACAUCUGGCUAAAAAGAA
1773
UUCUUUUUAGCCAGAUGUCAU
[2243-2263](21/21)


 


1274
GGGCUAGUUUCUGUGUAAGUG
1774
CACUUACACAGAAACUAGCCC
[2153-2173](21/21)


 


1275
UUUGGGCUAGUUUCUGUGUAA
1775
UUACACAGAAACUAGCCCAAA
[2150-2170](21/21)


 


1276
GGAGUGUCAGUAUGUUGAAUC
1776
GAUUCAACAUACUGACACUCC
[2088-2108](21/21)


 


1277
UGUGAUGUGAAAUGCUCAUAC
1777
GUAUGAGCAUUUCACAUCACA
[1995-2015](21/21)


 


1278
CUCAGCACCUUAUAUCUCGAA
1778
UUCGAGAUAUAAGGUGCUGAG
[1782-1802](21/21)


 


1279
UAAUUCGGGAUAUACGUAGGA
1779
UCCUACGUAUAUCCCGAAUUA
[1603-1623](21/21)


 


1280
AAAGACAAGAACAACUCCAAA
1780
UUUGGAGUUGUUCUUGUCUUU
[283-303](21/21)


 


1281
GAAAGACAAGAACAACUCCAA
1781
UUGGAGUUGUUCUUGUCUUUC
[282-302](21/21)


 


1282
CGAAAUGAUGUCCAAAGAGCA
1782
UGCUCUUUGGACAUCAUUUCG
[1559-1579](21/21)


 


1283
AACGAAAUGAUGUCCAAAGAG
1783
CUCUUUGGACAUCAUUUCGUU
[1557-1577](21/21)


 


1284
ACCUCCCUGUUGUUGACUUCA
1784
UGAAGUCAACAACAGGGAGGU
[1537-1557](21/21)


 


1285
AUCAUUAACCUCCCUGUUGUU
1785
AACAACAGGGAGGUUAAUGAU
[1530-1550](21/21)


 


1286
UUUCUGAUCUAUCACUUUGCA
1786
UGCAAAGUGAUAGAUCAGAAA
[1069-1089](21/21)


 


1287
CACUUGUUCCUGAUAUUCCCG
1787
CGGGAAUAUCAGGAACAAGUG
[538-558](21/21)


 


1288
GAUUUGAUUGACAUACUUUGG
1788
CCAAAGUAUGUCAAUCAAAUC
[165-185](21/21)


 


1289
ACGUUUCAGUCACUUGUUCCU
1789
AGGAACAAGUGACUGAAACGU
[528-548](21/21)


 


1290
ACAGGUGAAUUUCUCCCAAUU
1790
AAUUGGGAGAAAUUCACCUGU
[351-371](21/21)


 


1291
GAGCUGGUACUAAUAAAGGAU
1791
AUCCUUUAUUAGUACCAGCUC
[2372-2392](21/21)


 


1292
CUGUGAUGUGAAAUGCUCAUA
1792
UAUGAGCAUUUCACAUCACAG
[1994-2014](21/21)


 


1293
GAAAAACUAGAUUUAGGAGGA
1793
UCCUCCUAAAUCUAGUUUUUC
[1922-1942](21/21)


 


1294
GCUCAAAGAAAAAGGAGAAAA
1794
UUUUCUCCUUUUUCUUUGAGC
[1730-1750](21/21)


 


1295
AGACAAGAACAACUCCAAAAG
1795
CUUUUGGAGUUGUUCUUGUCU
[285-305](21/21)


 


1296
UUCGGGAUAUACGUAGGAGGG
1796
CCCUCCUACGUAUAUCCCGAA
[1606-1626](21/21)


 


1297
AUGUCCAAAGAGCAGUUCAAU
1797
AUUGAACUGCUCUUUGGACAU
[1566-1586](21/21)


 


1298
UGAUGUCCAAAGAGCAGUUCA
1798
UGAACUGCUCUUUGGACAUCA
[1564-1584](21/21)


 


1299
GAGCUGGAAAAACAGAAAAAA
1799
UUUUUUCUGUUUUUCCAGCUC
[252-272](21/21)


 


1300
CCGGCAUUUCACUAAACACAA
1800
UUGUGUUUAGUGAAAUGCCGG
[1144-1164](21/21)


 


1301
UUGAUGUUUCUGAUCUAUCAC
1801
GUGAUAGAUCAGAAACAUCAA
[1063-1083](21/21)


 


1302
CCAUUGAUGUUUCUGAUCUAU
1802
AUAGAUCAGAAACAUCAAUGG
[1060-1080](21/21)


 


1303
CCCAUUGAUGUUUCUGAUCUA
1803
UAGAUCAGAAACAUCAAUGGG
[1059-1079](21/21)


 


1304
GGCCCAUUGAUGUUUCUGAUC
1804
GAUCAGAAACAUCAAUGGGCC
[1057-1077](21/21)


 


1305
AAGAAGUAGGUAACUGUAGUC
1805
GACUACAGUUACCUACUUCUU
[823-843](21/21)


 


1306
GACCUUUUCUGAGCUAGUUUU
1806
AAAACUAGCUCAGAAAAGGUC
[1946-1966](21/21)


 


1307
AUCUCGAAGUUUUCAGCAUGC
1807
GCAUGCUGAAAACUUCGAGAU
[1795-1815](21/21)


 


1308
AAGACAAGAACAACUCCAAAA
1808
UUUUGGAGUUGUUCUUGUCUU
[284-304](21/21)


 


1309
GAAAACCCCAUUCACAAAAGA
1809
UCUUUUGUGAAUGGGGUUUUC
[1424-1444](21/21)


 


1310
GAUAUGGUACAACCCUUGUCA
1810
UGACAAGGGUUGUACCAUAUC
[1317-1337](21/21)


 


1311
GAAGUGGAAGAGCUAGAUAGU
1811
ACUAUCUAGCUCUUCCACUUC
[1242-1262](21/21)


 


1312
GUUUCUGAUCUAUCACUUUGC
1812
GCAAAGUGAUAGAUCAGAAAC
[1068-1088](21/21)


 


1313
GCCCAUUGAUGUUUCUGAUCU
1813
AGAUCAGAAACAUCAAUGGGC
[1058-1078](21/21)


 


1314
UUGCCCACAUUCCCAAAUCAG
1814
CUGAUUUGGGAAUGUGGGCAA
[427-447](21/21)


 


1315
GUUGCCCACAUUCCCAAAUCA
1815
UGAUUUGGGAAUGUGGGCAAC
[426-446](21/21)


 


1316
GCAGCAAACAAGAGAUGGCAA
1816
UUGCCAUCUCUUGUUUGCUGC
[1862-1882](21/21)


 


1317
UGCUCAAAGAAAAAGGAGAAA
1817
UUUCUCCUUUUUCUUUGAGCA
[1729-1749](21/21)


 


1318
GAAGCUCAACUUGCAUUAAUU
1818
AAUUAAUGCAAGUUGAGCUUC
[1587-1607](21/21)


 


1319
CAAAGAGCAGUUCAAUGAAGC
1819
GCUUCAUUGAACUGCUCUUUG
[1571-1591](21/21)


 


1320
GGAAAACCCCAUUCACAAAAG
1820
CUUUUGUGAAUGGGGUUUUCC
[1423-1443](21/21)


 


1321
UGAGCUGGAAAAACAGAAAAA
1821
UUUUUCUGUUUUUCCAGCUCA
[251-271](21/21)


 


1322
GCCCAGUAUCAGCAACAGCAU
1822
AUGCUGUUGCUGAUACUGGGC
[989-1009](21/21)


 


1323
UAGGUAACUGUAGUCCACAUU
1823
AAUGUGGACUACAGUUACCUA
[829-849](21/21)


 


1324
CAUUCCUGAGUUACAGUGUCU
1824
AGACACUGUAACUCAGGAAUG
[692-712](21/21)


 


1325
CCGUUUGUAGAUGACAAUGAG
1825
CUCAUUGUCAUCUACAAACGG
[495-515](21/21)


 


1326
GCUGGUACUAAUAAAGGAUUA
1826
UAAUCCUUUAUUAGUACCAGC
[2374-2394](21/21)


 


1327
AGGACACCAUUUGGGCUAGUU
1827
AACUAGCCCAAAUGGUGUCCU
[2141-2161](21/21)


 


1328
GCUCUCCAUAUCCCAUUCCCU
1828
AGGGAAUGGGAUAUGGAGAGC
[1500-1520](21/21)


 


1329
UGGGCCCAUUGAUGUUUCUGA
1829
UCAGAAACAUCAAUGGGCCCA
[1055-1075](21/21)


 


1330
ACUUUAAGCCAUUCACUCUCU
1830
AGAGAGUGAAUGGCUUAAAGU
[1023-1043](21/21)


 


1331
CACCUGCUACUUUAAGCCAUU
1831
AAUGGCUUAAAGUAGCAGGUG
[1015-1035](21/21)


 


1332
CUCACCUGCUACUUUAAGCCA
1832
UGGCUUAAAGUAGCAGGUGAG
[1013-1033](21/21)


 


1333
UGAAAAUGACAAGCUGGUUGA
1833
UCAACCAGCUUGUCAUUUUCA
[719-739](21/21)


 


1334
AGCUGGUACUAAUAAAGGAUU
1834
AAUCCUUUAUUAGUACCAGCU
[2373-2393](21/21)


 


1335
AUGCAAAAUCAUAGCCAAAAC
1835
GUUUUGGCUAUGAUUUUGCAU
[2031-2051](21/21)


 


1336
CAAACAAGAGAUGGCAAUGUU
1836
AACAUUGCCAUCUCUUGUUUG
[1866-1886](21/21)


 


1337
GAAAAUGACAAAAGCCUUCAC
1837
GUGAAGGCUUUUGUCAUUUUC
[1746-1766](21/21)


 


1338
AAAACCCCAUUCACAAAAGAC
1838
GUCUUUUGUGAAUGGGGUUUU
[1425-1445](21/21)


 


1339
AUGAGCUGGAAAAACAGAAAA
1839
UUUUCUGUUUUUCCAGCUCAU
[250-270](21/21)


 


1340
CUGAAGUGGAAGAGCUAGAUA
1840
UAUCUAGCUCUUCCACUUCAG
[1240-1260](21/21)


 


1341
ACUCCGGCAUUUCACUAAACA
1841
UGUUUAGUGAAAUGCCGGAGU
[1141-1161](21/21)


 


1342
CCAUUCACUCUCUGAACUUCU
1842
AGAAGUUCAGAGAGUGAAUGG
[1031-1051](21/21)


 


1343
AGCCAUUCACUCUCUGAACUU
1843
AAGUUCAGAGAGUGAAUGGCU
[1029-1049](21/21)


 


1344
CUGCUACUUUAAGCCAUUCAC
1844
GUGAAUGGCUUAAAGUAGCAG
[1018-1038](21/21)


 


1345
UCACCUGCUACUUUAAGCCAU
1845
AUGGCUUAAAGUAGCAGGUGA
[1014-1034](21/21)


 


1346
UGCCCUCACCUGCUACUUUAA
1846
UUAAAGUAGCAGGUGAGGGCA
[1009-1029](21/21)


 


1347
AUGCCCUCACCUGCUACUUUA
1847
UAAAGUAGCAGGUGAGGGCAU
[1008-1028](21/21)


 


1348
AUGGAAAAAGAAGUAGGUAAC
1848
GUUACCUACUUCUUUUUCCAU
[816-836](21/21)


 


1349
GAAGCCAAACUGACAGAAGUU
1849
AACUUCUGUCAGUUUGGCUUC
[762-782](21/21)


 


1350
UUAAAAAGCAUUGGAGUGUCA
1850
UGACACUCCAAUGCUUUUUAA
[2076-2096](21/21)


 


1351
GGCAAUGUUUUCCUUGUUCCC
1851
GGGAACAAGGAAAACAUUGCC
[1878-1898](21/21)


 


1352
ACAACUCAGCACCUUAUAUCU
1852
AGAUAUAAGGUGCUGAGUUGU
[1778-1798](21/21)


 


1353
GGCUGCUCAGAAUUGCAGAAA
1853
UUUCUGCAAUUCUGAGCAGCC
[1640-1660](21/21)


 


1354
AGCUCUCCAUAUCCCAUUCCC
1854
GGGAAUGGGAUAUGGAGAGCU
[1499-1519](21/21)


 


1355
AAGCUCUCCAUAUCCCAUUCC
1855
GGAAUGGGAUAUGGAGAGCUU
[1498-1518](21/21)


 


1356
AAGACAAACAUUCAAGCCGCU
1856
AGCGGCUUGAAUGUUUGUCUU
[1441-1461](21/21)


 


1357
AAACCCCAUUCACAAAAGACA
1857
UGUCUUUUGUGAAUGGGGUUU
[1426-1446](21/21)


 


1358
CGGCAUUUCACUAAACACAAG
1858
CUUGUGUUUAGUGAAAUGCCG
[1145-1165](21/21)


 


1359
CCCGUUUGUAGAUGACAAUGA
1859
UCAUUGUCAUCUACAAACGGG
[494-514](21/21)


 


1360
UCCCGUUUGUAGAUGACAAUG
1860
CAUUGUCAUCUACAAACGGGA
[493-513](21/21)


 


1361
AUCUGCCAACUACUCCCAGGU
1861
ACCUGGGAGUAGUUGGCAGAU
[407-427](21/21)


 


1362
GCCAGAUGUUAAGAAAAACUA
1862
UAGUUUUUCUUAACAUCUGGC
[1910-1930](21/21)


 


1363
AAAUGACAAAAGCCUUCACCU
1863
AGGUGAAGGCUUUUGUCAUUU
[1748-1768](21/21)


 


1364
CUGCUCAGAAUUGCAGAAAAA
1864
UUUUUCUGCAAUUCUGAGCAG
[1642-1662](21/21)


 


1365
AAGCUCAACUUGCAUUAAUUC
1865
GAAUUAAUGCAAGUUGAGCUU
[1588-1608](21/21)


 


1366
GUCAUCGGAAAACCCCAUUCA
1866
UGAAUGGGGUUUUCCGAUGAC
[1417-1437](21/21)


 


1367
CCCCAACCAGUUGACAGUGAA
1867
UUCACUGUCAACUGGUUGGGG
[899-919](21/21)


 


1368
GAAAAAGAAGUAGGUAACUGU
1868
ACAGUUACCUACUUCUUUUUC
[819-839](21/21)


 


1369
UGGAAAAAGAAGUAGGUAACU
1869
AGUUACCUACUUCUUUUUCCA
[817-837](21/21)


 


1370
CAAGUCCAGAAGCCAAACUGA
1870
UCAGUUUGGCUUCUGGACUUG
[754-774](21/21)


 


1371
UGGGAGGAGCUAUUAUCCAUU
1871
AAUGGAUAAUAGCUCCUCCCA
[675-695](21/21)


 


1372
UGCAAAACUAACCACUAUGUA
1872
UACAUAGUGGUUAGUUUUGCA
[2269-2289](21/21)


 


1373
AUGGAAAACCUUAUUCUCCUA
1873
UAGGAGAAUAAGGUUUUCCAU
[1828-1848](21/21)


 


1374
AGCCUUCACCUACUGAAAAAA
1874
UUUUUUCAGUAGGUGAAGGCU
[1758-1778](21/21)


 


1375
AAGGAGAAAAUGACAAAAGCC
1875
GGCUUUUGUCAUUUUCUCCUU
[1741-1761](21/21)


 


1376
AAAGACAAACAUUCAAGCCGC
1876
GCGGCUUGAAUGUUUGUCUUU
[1440-1460](21/21)


 


1377
UAAGUCCUGGUCAUCGGAAAA
1877
UUUUCCGAUGACCAGGACUUA
[1408-1428](21/21)


 


1378
UAUGAGCUGGAAAAACAGAAA
1878
UUUCUGUUUUUCCAGCUCAUA
[249-269](21/21)


 


1379
ACAACCCUUGUCACCAUCUCA
1879
UGAGAUGGUGACAAGGGUUGU
[1325-1345](21/21)


 


1380
GUACAACCCUUGUCACCAUCU
1880
AGAUGGUGACAAGGGUUGUAC
[1323-1343](21/21)


 


1381
GACACACUACUUGGCCUCAGU
1881
ACUGAGGCCAAGUAGUGUGUC
[1215-1235](21/21)


 


1382
CUGAUCUAUCACUUUGCAAAG
1882
CUUUGCAAAGUGAUAGAUCAG
[1072-1092](21/21)


 


1383
CCUGCUACUUUAAGCCAUUCA
1883
UGAAUGGCUUAAAGUAGCAGG
[1017-1037](21/21)


 


1384
CGAGAAGUAUUUGACUUCAGU
1884
ACUGAAGUCAAAUACUUCUCG
[213-233](21/21)


 


1385
UUCCCGUUUGUAGAUGACAAU
1885
AUUGUCAUCUACAAACGGGAA
[492-512](21/21)


 


1386
GCGCAGACAUUCCCGUUUGUA
1886
UACAAACGGGAAUGUCUGCGC
[483-503](21/21)


 


1387
UGGAUCUGCCAACUACUCCCA
1887
UGGGAGUAGUUGGCAGAUCCA
[404-424](21/21)


 


1388
GCUCAGUUACAACUAGAUGAA
1888
UUCAUCUAGUUGUAACUGAGC
[327-347](21/21)


 


1389
GCAAAAUCAUAGCCAAAACUA
1889
UAGUUUUGGCUAUGAUUUUGC
[2033-2053](21/21)


 


1390
CUGAGCUAGUUUUUUUGUACU
1890
AGUACAAAAAAACUAGCUCAG
[1954-1974](21/21)


 


1391
GGAGGAUUUGACCUUUUCUGA
1891
UCAGAAAAGGUCAAAUCCUCC
[1937-1957](21/21)


 


1392
UAGGAGGAUUUGACCUUUUCU
1892
AGAAAAGGUCAAAUCCUCCUA
[1935-1955](21/21)


 


1393
GCUGCUCAGAAUUGCAGAAAA
1893
UUUUCUGCAAUUCUGAGCAGC
[1641-1661](21/21)


 


1394
GUGGCUGCUCAGAAUUGCAGA
1894
UCUGCAAUUCUGAGCAGCCAC
[1638-1658](21/21)


 


1395
AAGUGGCUGCUCAGAAUUGCA
1895
UGCAAUUCUGAGCAGCCACUU
[1636-1656](21/21)


 


1396
UGGUCCUAAAACACCAGUACA
1896
UGUACUGGUGUUUUAGGACCA
[1286-1306](21/21)


 


1397
GAAUGGUCCUAAAACACCAGU
1897
ACUGGUGUUUUAGGACCAUUC
[1283-1303](21/21)


 


1398
GGCCUCAGUGAUUCUGAAGUG
1898
CACUUCAGAAUCACUGAGGCC
[1227-1247](21/21)


 


1399
UCUUCCAGCUAUGGAGACACA
1899
UGUGUCUCCAUAGCUGGAAGA
[1200-1220](21/21)


 


1400
GGCAUUUCACUAAACACAAGU
1900
ACUUGUGUUUAGUGAAAUGCC
[1146-1166](21/21)


 


1401
GACUCCGGCAUUUCACUAAAC
1901
GUUUAGUGAAAUGCCGGAGUC
[1140-1160](21/21)


 


1402
UGCUACUUUAAGCCAUUCACU
1902
AGUGAAUGGCUUAAAGUAGCA
[1019-1039](21/21)


 


1403
GUAUUUGACUUCAGUCAGCGA
1903
UCGCUGACUGAAGUCAAAUAC
[219-239](21/21)


 


1404
UGAGCCCAGUAUCAGCAACAG
1904
CUGUUGCUGAUACUGGGCUCA
[986-1006](21/21)


 


1405
GAGAAGUAUUUGACUUCAGUC
1905
GACUGAAGUCAAAUACUUCUC
[214-234](21/21)


 


1406
UCGAGAAGUAUUUGACUUCAG
1906
CUGAAGUCAAAUACUUCUCGA
[212-232](21/21)


 


1407
CAGUGAACUCAUUAAAUUCAG
1907
CUGAAUUUAAUGAGUUCACUG
[913-933](21/21)


 


1408
AGAAGCCAAACUGACAGAAGU
1908
ACUUCUGUCAGUUUGGCUUCU
[761-781](21/21)


 


1409
GGGAGGAGCUAUUAUCCAUUC
1909
GAAUGGAUAAUAGCUCCUCCC
[676-696](21/21)


 


1410
UUGGGAGGAGCUAUUAUCCAU
1910
AUGGAUAAUAGCUCCUCCCAA
[674-694](21/21)


 


1411
GCCCCUGUUGAUUUAGACGGU
1911
ACCGUCUAAAUCAACAGGGGC
[630-650](21/21)


 


1412
GACAGGUGAAUUUCUCCCAAU
1912
AUUGGGAGAAAUUCACCUGUC
[350-370](21/21)


 


1413
UGCAAAAUCAUAGCCAAAACU
1913
AGUUUUGGCUAUGAUUUUGCA
[2032-2052](21/21)


 


1414
UCCCAAAAGUAAGAAGCCAGA
1914
UCUGGCUUCUUACUUUUGGGA
[1895-1915](21/21)


 


1415
UUGUUCCCAAAAGUAAGAAGC
1915
GCUUCUUACUUUUGGGAACAA
[1891-1911](21/21)


 


1416
ACAACUCCAAAAGGAGCAAGA
1916
UCUUGCUCCUUUUGGAGUUGU
[293-313](21/21)


 


1417
UCCCAUUCCCUGUAGAAAAAA
1917
UUUUUUCUACAGGGAAUGGGA
[1510-1530](21/21)


 


1418
UCAUCGGAAAACCCCAUUCAC
1918
GUGAAUGGGGUUUUCCGAUGA
[1418-1438](21/21)


 


1419
CAGAGAAAGAAUUGCCUGUAA
1919
UUACAGGCAAUUCUUUCUCUG
[1390-1410](21/21)


 


1420
CAGAGCACUCACGUGCAUGAU
1920
AUCAUGCACGUGAGUGCUCUG
[1350-1370](21/21)


 


1421
CACCAGUACAUUCUUCUGGGG
1921
CCCCAGAAGAAUGUACUGGUG
[1297-1317](21/21)


 


1422
UCCGGCAUUUCACUAAACACA
1922
UGUGUUUAGUGAAAUGCCGGA
[1143-1163](21/21)


 


1423
UGACUCCGGCAUUUCACUAAA
1923
UUUAGUGAAAUGCCGGAGUCA
[1139-1159](21/21)


 


1424
CCCUCACCUGCUACUUUAAGC
1924
GCUUAAAGUAGCAGGUGAGGG
[1011-1031](21/21)


 


1425
GUCCAGAAGCCAAACUGACAG
1925
CUGUCAGUUUGGCUUCUGGAC
[757-777](21/21)


 


1426
GCUGGUUGAGACUACCAUGGU
1926
ACCAUGGUAGUCUCAACCAGC
[731-751](21/21)


 


1427
UUCCUGAGUUACAGUGUCUUA
1927
UAAGACACUGUAACUCAGGAA
[694-714](21/21)


 


1428
CCCCUGUUGAUUUAGACGGUA
1928
UACCGUCUAAAUCAACAGGGG
[631-651](21/21)


 


1429
CCAGUGGAUCUGCCAACUACU
1929
AGUAGUUGGCAGAUCCACUGG
[400-420](21/21)


 


1430
AGACAGGUGAAUUUCUCCCAA
1930
UUGGGAGAAAUUCACCUGUCU
[349-369](21/21)


 


1431
UCGCUCAGUUACAACUAGAUG
1931
CAUCUAGUUGUAACUGAGCGA
[325-345](21/21)


 


1432
CCAGAUGUUAAGAAAAACUAG
1932
CUAGUUUUUCUUAACAUCUGG
[1911-1931](21/21)


 


1433
AGCCAGAUGUUAAGAAAAACU
1933
AGUUUUUCUUAACAUCUGGCU
[1909-1929](21/21)


 


1434
AUGGCAAUGUUUUCCUUGUUC
1934
GAACAAGGAAAACAUUGCCAU
[1876-1896](21/21)


 


1435
AGAGAUGGCAAUGUUUUCCUU
1935
AAGGAAAACAUUGCCAUCUCU
[1872-1892](21/21)


 


1436
GAACAACUCCAAAAGGAGCAA
1936
UUGCUCCUUUUGGAGUUGUUC
[291-311](21/21)


 


1437
GGGCAAAAGCUCUCCAUAUCC
1937
GGAUAUGGAGAGCUUUUGCCC
[1492-1512](21/21)


 


1438
AUCGGAAAACCCCAUUCACAA
1938
UUGUGAAUGGGGUUUUCCGAU
[1420-1440](21/21)


 


1439
ACGUGCAUGAUGCCCAAUGUG
1939
CACAUUGGGCAUCAUGCACGU
[1360-1380](21/21)


 


1440
GGUCCUAAAACACCAGUACAU
1940
AUGUACUGGUGUUUUAGGACC
[1287-1307](21/21)


 


1441
CAGUCAGCGACGGAAAGAGUA
1941
UACUCUUUCCGUCGCUGACUG
[230-250](21/21)


 


1442
CUCCGGCAUUUCACUAAACAC
1942
GUGUUUAGUGAAAUGCCGGAG
[1142-1162](21/21)


 


1443
GACUUCAGUCAGCGACGGAAA
1943
UUUCCGUCGCUGACUGAAGUC
[225-245](21/21)


 


1444
AAGCCAUUCACUCUCUGAACU
1944
AGUUCAGAGAGUGAAUGGCUU
[1028-1048](21/21)


 


1445
CUGAGCCCAGUAUCAGCAACA
1945
UGUUGCUGAUACUGGGCUCAG
[985-1005](21/21)


 


1446
GACAGUGAACUCAUUAAAUUC
1946
GAAUUUAAUGAGUUCACUGUC
[911-931](21/21)


 


1447
GAAGACCCCAACCAGUUGACA
1947
UGUCAACUGGUUGGGGUCUUC
[894-914](21/21)


 


1448
AGCCAAACUGACAGAAGUUGA
1948
UCAACUUCUGUCAGUUUGGCU
[764-784](21/21)


 


1449
AAGCUGGUUGAGACUACCAUG
1949
CAUGGUAGUCUCAACCAGCUU
[729-749](21/21)


 


1450
GAGGAGCUAUUAUCCAUUCCU
1950
AGGAAUGGAUAAUAGCUCCUC
[678-698](21/21)


 


1451
GGUCCACAGCUCAUCAUGAUG
1951
CAUCAUGAUGAGCUGUGGACC
[99-119](21/21)


 


1452
GCUACUAAUCAGGCUCAGUCA
1952
UGACUGAGCCUGAUUAGUAGC
[585-605](21/21)


 


1453
CCCAGUCUUCAUUGCUACUAA
1953
UUAGUAGCAAUGAAGACUGGG
[572-592](21/21)


 


1454
UGAUGACUGCAUGCAGCUUUU
1954
AAAAGCUGCAUGCAGUCAUCA
[461-481](21/21)


 


1455
GAGACAGGUGAAUUUCUCCCA
1955
UGGGAGAAAUUCACCUGUCUC
[348-368](21/21)


 


1456
UUUCGCUCAGUUACAACUAGA
1956
UCUAGUUGUAACUGAGCGAAA
[323-343](21/21)


 


1457
AUGAUAUGACAUCUGGCUAAA
1957
UUUAGCCAGAUGUCAUAUCAU
[2238-2258](21/21)


 


1458
GGACACCAUUUGGGCUAGUUU
1958
AAACUAGCCCAAAUGGUGUCC
[2142-2162](21/21)


 


1459
UCAUAGCCAAAACUAGUAUAG
1959
CUAUACUAGUUUUGGCUAUGA
[2039-2059](21/21)


 


1460
UCUGAGCUAGUUUUUUUGUAC
1960
GUACAAAAAAACUAGCUCAGA
[1953-1973](21/21)


 


1461
GAGAUGGCAAUGUUUUCCUUG
1961
CAAGGAAAACAUUGCCAUCUC
[1873-1893](21/21)


 


1462
GUGAAUACUCCCUGCAGCAAA
1962
UUUGCUGCAGGGAGUAUUCAC
[1849-1869](21/21)


 


1463
AACCUUAUUCUCCUAGUGAAU
1963
AUUCACUAGGAGAAUAAGGUU
[1834-1854](21/21)


 


1464
GGAAAACCUUAUUCUCCUAGU
1964
ACUAGGAGAAUAAGGUUUUCC
[1830-1850](21/21)


 


1465
GCCUUCACCUACUGAAAAAAC
1965
GUUUUUUCAGUAGGUGAAGGC
[1759-1779](21/21)


 


1466
UGCUCAGAAUUGCAGAAAAAG
1966
CUUUUUCUGCAAUUCUGAGCA
[1643-1663](21/21)


 


1467
GAGUAUGAGCUGGAAAAACAG
1967
CUGUUUUUCCAGCUCAUACUC
[246-266](21/21)


 


1468
AUGGUCCUAAAACACCAGUAC
1968
GUACUGGUGUUUUAGGACCAU
[1285-1305](21/21)


 


1469
ACACACUACUUGGCCUCAGUG
1969
CACUGAGGCCAAGUAGUGUGU
[1216-1236](21/21)


 


1470
GAGACACACUACUUGGCCUCA
1970
UGAGGCCAAGUAGUGUGUCUC
[1213-1233](21/21)


 


1471
AUGGAGACACACUACUUGGCC
1971
GGCCAAGUAGUGUGUCUCCAU
[1210-1230](21/21)


 


1472
AGCUAUGGAGACACACUACUU
1972
AAGUAGUGUGUCUCCAUAGCU
[1206-1226](21/21)


 


1473
GCAUUUCACUAAACACAAGUC
1973
GACUUGUGUUUAGUGAAAUGC
[1147-1167](21/21)


 


1474
UCAGUCAGCGACGGAAAGAGU
1974
ACUCUUUCCGUCGCUGACUGA
[229-249](21/21)


 


1475
AUUCACUCUCUGAACUUCUAA
1975
UUAGAAGUUCAGAGAGUGAAU
[1033-1053](21/21)


 


1476
AACACAGAUUUUGGUGAUGAA
1976
UUCAUCACCAAAAUCUGUGUU
[945-965](21/21)


 


1477
GUCAACACAGAUUUUGGUGAU
1977
AUCACCAAAAUCUGUGUUGAC
[942-962](21/21)


 


1478
GAACUCAUUAAAUUCAGAUGC
1978
GCAUCUGAAUUUAAUGAGUUC
[917-937](21/21)


 


1479
ACCAGUUGACAGUGAACUCAU
1979
AUGAGUUCACUGUCAACUGGU
[904-924](21/21)


 


1480
AGGUAACUGUAGUCCACAUUU
1980
AAAUGUGGACUACAGUUACCU
[830-850](21/21)


 


1481
UAUACCCUCAAUGGAAAAAGA
1981
UCUUUUUCCAUUGAGGGUAUA
[806-826](21/21)


 


1482
UCCAUUCCUGAGUUACAGUGU
1982
ACACUGUAACUCAGGAAUGGA
[690-710](21/21)


 


1483
GGAGCUAUUAUCCAUUCCUGA
1983
UCAGGAAUGGAUAAUAGCUCC
[680-700](21/21)


 


1484
ACGGUAUGCAACAGGACAUUG
1984
CAAUGUCCUGUUGCAUACCGU
[646-666](21/21)


 


1485
CCCUGUUGAUUUAGACGGUAU
1985
AUACCGUCUAAAUCAACAGGG
[632-652](21/21)


 


1486
GUAGCCCCUGUUGAUUUAGAC
1986
GUCUAAAUCAACAGGGGCUAC
[627-647](21/21)


 


1487
GAAACUUCUGUUGCUCAGGUA
1987
UACCUGAGCAACAGAAGUUUC
[609-629](21/21)


 


1488
GAGAGCCCAGUCUUCAUUGCU
1988
AGCAAUGAAGACUGGGCUCUC
[567-587](21/21)


 


1489
AAACCAGUGGAUCUGCCAACU
1989
AGUUGGCAGAUCCACUGGUUU
[397-417](21/21)


 


1490
UUAGGAGGAUUUGACCUUUUC
1990
GAAAAGGUCAAAUCCUCCUAA
[1934-1954](21/21)


 


1491
AAGCCAGAUGUUAAGAAAAAC
1991
GUUUUUCUUAACAUCUGGCUU
[1908-1928](21/21)


 


1492
UUCCCAAAAGUAAGAAGCCAG
1992
CUGGCUUCUUACUUUUGGGAA
[1894-1914](21/21)


 


1493
GGAGCAAGAGAAAGCCUUUUU
1993
AAAAAGGCUUUCUCUUGCUCC
[305-325](21/21)


 


1494
GAUGGCAAUGUUUUCCUUGUU
1994
AACAAGGAAAACAUUGCCAUC
[1875-1895](21/21)


 


1495
AAGGAGCAAGAGAAAGCCUUU
1995
AAAGGCUUUCUCUUGCUCCUU
[303-323](21/21)


 


1496
AAGCCUUCACCUACUGAAAAA
1996
UUUUUCAGUAGGUGAAGGCUU
[1757-1777](21/21)


 


1497
UGAAGCUCAACUUGCAUUAAU
1997
AUUAAUGCAAGUUGAGCUUCA
[1586-1606](21/21)


 


1498
AUGAAGCUCAACUUGCAUUAA
1998
UUAAUGCAAGUUGAGCUUCAU
[1585-1605](21/21)


 


1499
CCCAUUCCCUGUAGAAAAAAU
1999
AUUUUUUCUACAGGGAAUGGG
[1511-1531](21/21)


 


1500
UUGGAGGCUCAUCUCACAAGA
2000
UCUUGUGAGAUGAGCCUCCAA
[1461-1481](21/21)


 


1501
AGCUGGAAAAACAGAAAAAAC
2001
GUUUUUUCUGUUUUUCCAGCU
[253-273](21/21)


 


1502
GUGCAUGAUGCCCAAUGUGAG
2002
CUCACAUUGGGCAUCAUGCAC
[1362-1382](21/21)


 


1503
CACGUGCAUGAUGCCCAAUGU
2003
ACAUUGGGCAUCAUGCACGUG
[1359-1379](21/21)


 


1504
ACACCAGUACAUUCUUCUGGG
2004
CCCAGAAGAAUGUACUGGUGU
[1296-1316](21/21)


 


1505
GUCCUAAAACACCAGUACAUU
2005
AAUGUACUGGUGUUUUAGGAC
[1288-1308](21/21)


 


1506
UGGAGACACACUACUUGGCCU
2006
AGGCCAAGUAGUGUGUCUCCA
[1211-1231](21/21)









Example 2
In Vitro Testing of siRNA Compounds for Nrf2
1. General
1.5-2×105 tested cells (HeLa or 293 cells) were seeded per well in a 6-well plate (70-80% confluent).
24 h subsequently, cells were transfected with siRNA oligos using lipofectamine 2000 reagent (Invitrogene) at a final concentration of 500 pM, 5 nM, 20 nM or 40 nM. The cells were incubated at 37° C. in a CO2 incubator for 72 h.
As positive control for cell transfection, PTEN-Cy3 labeled siRNA oligos were used.
As negative control for siRNA activity, GFP siRNA oligos were used.
72 h after transfection cells were harvested and RNA was extracted from cells.
Transfection efficiency was tested by fluorescent microscopy.
Results:
The percent of inhibition of gene expression using specific preferred siRNAs was determined using qPCR analysis of target gene in cells expressing the endogenous gene. The data in Table C demonstrate the percent of knockdown of the expression of the target gene in cells. In general, the siRNAs having specific sequences that were selected for in vitro testing were specific for both human and the rat/rabbit genes. Similar results of reduced expression of specific genes are obtained with other siRNAs, the sequences of which are listed in Tables A and B.








TABLE C







Percent of knockdown of the expression of the target Nrf2



human gene in cells using siRNAs having preferred sequences


(results of separate experiments are indicated).












% knockdown of



Active siRNA
Sequence
the human gene





NFE2L2_10
Sense:
20 nM in HeLa cells:



(Sense SEQ ID NO: 501)
CCCUGUCGAAAAAAUCAUU
91%, 54%, 51%


having one mismatch
Antisense:
inhibition


(in bold) compared to
AAUGAUUUUUUCGACAGGG
5 nM in HeLa cells:


human sequence

91% inhibition


 


NFE2L2_4
Sense:
20 nM 80%, 41%


(Sense SEQ ID NO: 119)
CCCAUUCACAAAAGACAAA
inhibition



Antisense:
5 nM-73%, 36%



UUUGUCUUUUGUGAAUGGG
inhibition


 


NFE2L2_5
Sense:
20 nM-69%, 17%


(Sense SEQ ID NO: 195):
CAGCAGGACAUGGAUUUGA
inhibition



Antisense:
5 nM-47%, 16%



UCAAAUCCAUGUCCUGCUG
inhibition


 


NFE2L2_9
Sense:
20 nM-44%


(Sense SEQ ID NO: 502)
UCCCUGUCGAAAAAAUCAU
inhibition



Antisense:
5 nM-25%



AUGAUUUUUUCGACAGGGA
inhibition


 


NFE2L2_11
Sense:
20 nM-46%


(Sense SEQ ID NO: 503)
CCUGUCGAAAAAAUCAUUA
inhibition



Antisense:
5 nM-23%



UAAUGAUUUUUUCGACAGG
inhibition


 


NFE2L2_1
Sense:
20 nM-55%, 73%,


(Sense SEQ ID NO: 2)
GGAGGGGUAAGAAUAAAGU
53% inhibition



Antisense:



ACUUUAUUCUUACCCCUCC


 


NFE2L2_2
Sense:
20 nM-92%, 49%,


(Sense SEQ ID NO: 7)
GCCCUCACCUGCUACUUUA
46% inhibition



Antisense:



UAAAGUAGCAGGUGAGGGC


 


NFE2L2_3
Sense:
20 nM-96%, 83%,


(Sense SEQ ID NO: 10)
UCCCGUUUGUAGAUGACAA
82% inhibition



Antisense:



UUGUCAUCUACAAACGGGA


 


NFE2L2_12
Sense:
20 nM-95%, 96%,


(Sense SEQ ID NO: 26)
GUAAGAAGCCAGAUGUUAA
76% inhibition



Antisense:



UUAACAUCUGGCUUCUUAC









Example 3
The Effect of Nrf2 siRNA Treatment on Tumor Growth in Vivo
Methods:
Tumor Xenografts: A549 cells (5×106) were injected into the hind leg of male athymic nude mice and the tumor was measured weekly. The tumor volumes were measured using the following formula: [length (mm)×width (mm)×width (mm)×0.52]. In the lung metastasis experiments, 2×106 A549-C8-luc cells were injected into SCID-Beige mice (Charles River, Mass.) intravenously.
For the in vivo experiments, all siRNA compounds were chemically synthesized being stabilized by alternating 2-O′-Me modifications. The sequence of siRNA targeting human Nrf2 used for in vivo experiments is 5′-UCCCGUUUGUAGAUGACAA-3′ (sense, SEQ ID NO:10) and 5′-UUGUCAUCUACAAACGGGA-3′ (antisense, SEQ ID NO:513). The sequence of control siRNA targeting GFP is 5′-GGCUACGUCCAGGAGCGCACC-3′ (sense, SEQ ID NO: 2007) and 5′-GGUGCGCUCCUGGACGUAGCC-3′ (antisense, SEQ ID NO: 2008).
For in vivo delivery of siRNA into subcutaneous tumors, siRNA duplexes diluted in PBS were injected into the hind leg tumors using insulin syringes at a concentration of 10 μg/mm3. Intraperitoneal injections of carboplatin were given at a dose of 40 mg/kg body weight. Both siRNA and carboplatin were administered twice weekly for 4 weeks.
For lung tumor delivery, female C57B6 mice were injected with Lewis Lung Carcinoma (LLC) cells (0.5×106) intravenously, 24 days prior to the delivery experiment. Upon development of lung metastases, mice were administered with 100 μg/mouse of Cy3-labeled naked chemically stabilized reference siRNA via nebulizer inhalation on 3 consequent days. Mice were euthanized 24 hrs after the last inhalation. Upon termination, lungs were inflated with ice-cold 4% paraformaldehyde, followed by manual sectioning with razor blades. Clearly visible large surface tumors were sectioned separately. Resulting sections were analyzed by Bio-Rad Confocal microscope using a 20× Water objective and 2× zoom combined to give a total of 40× magnification. Control, non-siRNA-treated lungs were used to set up background fluorescence level.
For aerosol delivery of Nrf2 or GFP siRNA into lung tumors, 100 μg of siRNA duplex diluted in PBS was aerosolized using a nebulizer. Mice were given three dose of siRNA (100 μg/dose) every week, for 4 weeks, using a nebulizer. Intraperitoneal injections of carboplatin were given at a dose of 30 mg/kg body weight twice/week.
In Vivo Imaging: Animals inoculated with A549-C8-luc cells, which express a luciferase reporter gene, were anesthetized and injected intraperitoneally with 250 ul of luminescent substrate (15 mg/ml stock) D-Luciferin Firefly (Xenogen Cat# XR-1001). The animals were then imaged and analyzed by using the Xenogen IVIS Optical Imaging Device in the Johns Hopkins Oncology Center.
Statistical Analysis-Statistical comparisons were performed by Student's t-tests. A value of p<0.05 was considered statistically significant. Tumor weights and changes in tumor volume were summarized using descriptive statistics. Differences in tumor measures between treatment groups were examined using linear regression models with generalized estimating equations (GEE). The distributions of both tumor measurements were skewed, so log transformations were used.
Results:
A tumor xenograft experiment with A549 cells which were injected into the hind leg was conducted in order to test the anti-tumor activity of Nrf2 siRNA in vivo. Mice bearing subcutaneous tumors were treated with Nrf2 siRNA (comprising SEQ ID NO: 10) by direct injection into the tumor and by carboplatin twice a week for 4 weeks and tumor weight was measured at the termination of the experiment. Treatment with control non-targeting siRNA (GFP siRNA) did not inhibit tumor growth as compared to control mice treated with PBS alone. The tumor weights were significantly different between GFP siRNA and Nrf2 siRNA treated tumors (P=0.0002). Treatment with Nrf2 siRNA alone reduced mean tumor weight by 53% (±20% SD) compared to the control group. When Nrf2 siRNA was combined with carboplatin, there was an even greater reduction in mean tumor weight in all animals. To explore the effect of radiation exposure in combination with Nrf2 inhibition in vivo, the same A549 cell tumor xenograft model was used. In comparison with control siRNA+ionizing radiation (IR) treated tumors, combination of Nrf2 siRNA+IR (2 fractions of 3Gy irradiation) produced an additive effect on tumor growth inhibition (Tables D1 and D2). Delivery of naked Nrf2 siRNA duplex into tumor inhibited the expression of Nrf2 and its downstream target genes (HO-1 and GCLm), P<0.05 (FIG. 1).







TABLE D1







Mean (SD) of tumor weights and changes in tumor volume


in tumor xenograft mice following treatment with Nrf2 siRNA


(SEQ ID NO: 10), carboplatin, radiation and combination


thereof (Experiment 1).












Mean (SD) change in
Mean (SD) tumor



Treatment
tumor volume (mm3)
weight (mg)







GFP siRNA
532.94 (260.94)
 426 (187.83)



GFP siRNA +
246.77 (189.39)
341.25 (275.27)  



radiation



GFP siRNA +
352.34 (170.96)
212 (66.48)



carboplatin



Nrf2 siRNA
249.17 (111.37)
238 (64.58)



Nrf2 siRNA +
116.04 (147.9) 
118 (70.85)



radiation



Nrf2 siRNA +
58.78 (80.06)
110 (57.62)



carboplatin

















TABLE D2







Mean (SD) of tumor weights and changes in tumor volume


in tumor xenograft mice following treatment with Nrf2 siRNA


(SEQ ID NO: 10), carboplatin, radiation and combination


thereof (Experiment 2).












Mean (SD) change in
Mean (SD) tumor



Treatment
tumor volume (mm3)
weight (mg)

















GFP siRNA
375
(233.7)
327.5
(201.56)



GFP siRNA +
187.33
(76.66)
185
(67.16)



carboplatin



Nrf2 siRNA
138.67
(128.24)
143.33
(90.74)



Nrf2 siRNA +
45
(33.81)
58.33
(21.37)



carboplatin










The delivery of naked siRNA duplexes into orthotopic lung tumors was examined. Mice were injected with Lewis lung carcinoma cells and 24 days later (when the mice developed larger tumors) mice were inhaled for three consecutive days with 100 μg/day/mouse of Cy3 labeled naked chemically stabilized reference siRNA using a nebulizer. Twenty four hours after last siRNA administration, mice were sacrificed; lungs harvested and sectioned. Resulting sections were analyzed by Bio-Rad Confocal microscope using a 20× Water objective and 2× zoom combined to give a total of 40× magnification. Control, non-siRNA-treated lungs were used to set up background fluorescence level. The fluorescence results revealed localization of Cy3 labeled siRNA in a large surface tumor, especially in intraparenchymal tumor. The large surface-protruding tumors showed Cy3 signal but the intensity was several folds lower than that observed in the small intra-parenchymal tumors.
In the next step, mice with orthotopic growing tumors expressing ARE-Luc Nrf2-dependent reporter were used. The mice with A549 lung tumors were treated with Nrf2 siRNA (SEQ ID NO: 10) intranasally using a nebulizer followed by carboplatin treatment. As demonstrated in FIG. 2, Nrf2 siRNA administered intranasally inhibited Nrf2 reporter activity in vivo, indicating specific inhibition of Nrf2 expression using Nrf2 siRNA following intranasal administration. Mice receiving Nrf2 siRNA together with carboplatin demonstrated higher growth inhibition as compared with control mice receiving GFP siRNA together with carboplatin (Table D3). The mean lung weight at termination and the luminescent flux intensity (evaluated by in vivo imaging) were lowest in mice treated with Nrf2 siRNA+carboplatin. Thus, combination of Nrf2 siRNA with carboplatin/radiation led to a reduction in tumor growth after 4 weeks of treatment compared with either agent alone. This study suggests that Nrf2 siRNA inhibitors may be considered as highly efficient promoters for the antineoplastic potential of platinum drugs such as carboplatin, causing additive/synergistic effects in lung cancer cells.







TABLE D3







Mean (SD) of lung tumor weights following intranasal treatment


with Nrf2 siRNA (SEQ ID NO: 10) alone, and in combination


with carboplatin.










Treatment
Mean (SD) tumor weight (mg)







GFP siRNA
 710 (167.93)



GFP siRNA +
480 (95.13)



carboplatin



Nrf2 siRNA
 802 (172.25)



Nrf2 siRNA +
370 (62.05)



carboplatin



Vehicle siRNA
760 (98.99)



Vehicle siRNA +
495 (49.5) 



carboplatin

## Claims

1. A double-stranded siRNA compound having the structure:










5′    (N)x-Z 3′
(antisense strand)



 



3′ Z′-(N′)y   5′
(sense strand)





wherein each N and N′ is a ribonucleotide which may independently be modified or unmodified in its sugar residue;
wherein each of (N)x and (N′)y is an oligomer in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond;
wherein each of x and y is an integer between 19 and 40;
wherein each of Z and Z′ may be present or absent, but if present is 1-5 consecutive nucleotides covalently attached at the 3′ terminus of the strand in which it is present;
and wherein the sequence of (N)x comprises SEQ ID NO: 513 and the sequence of (N′)y comprises SEQ ID NO: 10 which is complementary to SEQ ID NO: 513.

2. The compound of claim 1, wherein the covalent bond is a phosphodiester bond.

3. The compound of claim 1, wherein x=y=19.

4. The compound of claim 3, wherein both Z and Z′ are absent.

5. The compound of claim 3, wherein one of Z or Z′ is present.

6. The compound of claim 1, wherein all of the ribonucleotides in (N)x or (N′)y or both are unmodified in their sugar residues.

7. The compound of claim 1, wherein at least one ribonucleotide in (N)x or (N′)y or both comprises a modification in its sugar residue.

8. The compound of claim 7, wherein the modification comprises a modification at the 2′ position of the sugar residue.

9. The compound of claim 8, wherein the modification at the 2′ position of the sugar residue comprises the presence of a moiety selected from the group comprising amino, fluoro, alkoxy and alkyl groups.

10. The compound according to claim 9, wherein the modification at the 2′ position of the sugar residue comprises the presence of an alkoxy group.

11. The compound of claim 10, wherein the alkoxy group is a methoxy (2′-O-methyl) group.

12. The compound of claim 11, wherein alternating ribonucleotides in the antisense strand or the sense strand or both strands are modified in their sugar residues.

13. The compound of claim 12, wherein each ribonucleotide at the 5′ and 3′ termini of the antisense strand is modified in its sugar residues, and each ribonucleotide at the 5′ and 3′ termini of the sense strand is unmodified in its sugar residues.

14. The compound of claim 13, wherein the antisense and the sense strands are non-phosphorylated at the 3′ and 5′ termini or wherein both the antisense and the sense strands are phosphorylated at the 3′ termini.

15. A composition comprising a pharmaceutically acceptable carrier and a compound of claim 1.

16. A method of treating a patient suffering from a cancerous disease comprising administering to the patient the compound of claim 1 in an amount effective to treat the patient.

17. The method of claim 16, wherein the cancerous disease is selected from the group consisting of lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer.

18. The method of claim 17, wherein the cancerous disease is a lung cancer.

19. The method of claim 18, wherein the compound is a naked siRNA compound.

20. The method of claim 19, wherein the composition is administered via aerosol directly to the inner lung of the patient.

21. The method of claim 16, further comprising administering to the patient a chemotherapy drug.

22. The method of claim 21, wherein the chemotherapy drug is a platinum drug.

23. A double-stranded siRNA compound comprising the structure:



wherein each of A, C, U, and G is an unmodified or a 2′-O-methyl sugar-modified ribonucleotide and each consecutive ribonucleotide is joined to the next ribonucleotide by a covalent bond, and
wherein each vertical line represents base pairing between the ribonucleotides at either end of the line.

24. The method of claim 18, wherein the lung cancer is non-small cell lung carcinoma or small cell lung carcinoma.