CN101355970A - Peptide-DICER substrate RNA conjugates as delivery vehicles for SIRNA - Google Patents

Abstract

Provided are compositions comprising a double stranded ribonucleic acid (dsRNA) molecule and a peptide of about 5 to about 40 amino acids, wherein the dsRNA molecule is conjugated to the peptide. The strands of the dsRNA may have lengths from about 25 to about 30 base pairs, which may be the same or different. siRNA may, alternatively, comprise at least three strands (i.e., either at least two sense strands and one antisense strand or at least two antisense strands and one sense strand) wherein the at least two sense strands or the at least two antisense strands are separated by a nick or a gap of at least one nucleotide.

Description

Peptide-DICER substrate RNA conjugates as the SIRNA delivery vehicle

Background of invention

Technical field

The present invention relates generally to disturb (RNAi) to treat illness by RNA.More particularly, the present invention relates to little inhibition nucleic acid (siRNA) molecule and the targeted delivery of variant thereof, this siRNA and variant thereof can mediate rna i antagonism expression of gene.But siRNA yoke described here is bonded to one or more peptides, and wherein the peptide that closes of yoke helps to send, and improves stability, and/or reduces the toxicity of siRNA.

Description of related art

With delivery of nucleic acids is a free-revving engine of molecular biology and clinical research to animal and human (plant).More particularly, current development in gene therapy, antisense therapy and RNA interference (RNAi) therapy field has produced the needs that the more effective ways of cell are introduced nucleic acid in exploitation.

So far, the common methods of delivery of nucleic acids to the cell used many methods of cationic lipid, electroporation and film rupture virus transduction and application machine or biochemical and/or infiltration (for example, using detergent, microinjection or particle gun).These methods are perplexed by various shortcoming.For example, although the most normal DNA of being used to of cation lipid and external the sending of siRNA (siRNA), their common toxicity is big, therefore is unsuitable for using in the body such as the treatment disease.As for the virus transduction, there is such probability, promptly replying as the replication defective virus of delivery vehicle is wild type, thereby has become morbific.Electroporation is endured the relatively poor gene transfering efficiency and the influence of cell injury to the fullest extent, has therefore limited clinical practice.

RNA disturbs (RNAi) to occur as a kind of promising technology, the expression of specific gene that is used for modified plant and zooblast, therefore expect that it provides the therapy of usefulness (instrument), use with treatment and modify various diseases or the illness that endogenous gene expression can be treated.RNA disturbs the process be meant in animal with short RNA interfering (siRNA) mediation sequence specific post transcriptional gene silencing, this siRNA normally sequence homology in the dsRNA of part targeting messenger RNA (mRNA).Referring to Fire etc., Nature 391:806,1998 and Hamilton etc., Science 286:950-951,1999.Respective process in plant is commonly referred to as PTGS or RNA silence, is known as oppressive (quelling) in fungus.The process of PTGS is considered to the conservative cytophylaxis mechanism of a kind of evolution property, is used to stop the expression of alien gene, and is shared (Fire etc., Trends Genet, 15:358,1999) by different floras and door usually.

Suitable dsRNA is introduced the destruction that causes endogenous, mRNA of the same clan (that is, be introduced into dsRNA share the mRNA that sequence in fact is equal to) in the cell.The mechanism of the mediated targeted gene silencing of dsRNA duplex is considered to the process of two steps.At first, dsRNA by rnase iii enzyme～be known as dicer～be fractured into (being degraded to) little RNA interfering (siRNA) (Hamilton etc., the same; Berstein etc., Nature 409:363,2001).Derived from the active short interfering rna of dicer mainly be have two bases 3 ' dangle, length be about 21 to about 23 nucleotide (Kim etc., Nature Biotech.23 (2): 222,2005).Recent evidence shows, long dsRNA (length is 25-30 nucleotide) is comparable to be oriented to identical target spot, their shorter counterpart (21-mer) has bigger active for gene silencing (Kim etc., Nature Biotech.23 (2): 222,2005).In addition, for for 21-mer RNA mediation siRNA gene silencing, being some obstinate target spot, can use longer 27-mer siRNA duplex silence effectively.It is substrate (Kim etc., the Nature Biotech.23 (2): 222,2005) of Dicer Cobra venom endonuclease that the usefulness of these longer siRNA duplexs improves owing to them.Therefore, aspect therapeutic agent, these longer dsRNA play the effect of precursor, and when adding man-hour with dicer, it enters the function of RISC complex and the reticent amboceptor of performance target gene.

Second step comprised siRNA mixed and is known as in the multicomponent nucleic acid enzymes complex that RNA induces silencing complex or " RISC ".By they complementarity to siRNA duplex antisense strand, RISC discerns the mRNA substrate, and realizes the silence of gene expression by the destruction (cracking) that is attached to and starts targeting mRNA.The cracking of target RNA occurs in the middle part, zone (Elbashir etc., Genes Dev.15:188,2001) that is complementary to siRNA duplex antisense strand.

The needs of the better tool and method of long-term existence in the art so that nucleic acid, peptide and other pharmacological agent are delivered to cell, particularly are subjected to the relatively poor efficient of delivery of agents in view of existing cell delivery feed technique and/or toxicity is big limits.There are the relevant needs of improving one's methods with preparation, so that send effective dose with active and persistent state, and thereby application can change the phenotype or the morbid state of targeted cells like this to the adjusting of selected cell, tissue or the expression of the avirulent delivery medium body of inner chamber mediated gene.

Summary of the invention

The present invention has solved these and other relevant needs by providing the polypeptide/dicer substrate dsRNA conjugates that particularly improves polynucleotide and send to be used as treating treatment of diseases prodrug delivery system.After polypeptide delivery was to the cell, precursor siRNA was discharged it by the dicer enzyme action from send peptide, allow siRNA to enter RISC complex and target-specific gene so subsequently, is used for PTGS.Peptide as described herein-dsRNA conjugates provides a kind of promising new method, is used for improving dsRNA therapeutic precursor and enters sending of cell, is used to modify the extensive disease that endogenous gene expression can treat and the treatment of illness.The polypeptide that raising polynucleotide as described herein are sent/dicer substrate dsRNA conjugates has advantageously improved protection and/or the preventative ability of dsRNA by the volume lifetime that prolongs it.

On the one hand, the invention provides compositions, comprise pharmaceutical composition, it is suitable for animal is used one or more double stranded RNAs (dsRNA) molecule, wherein said compositions comprises one or more dsRNA molecules and one or more peptides, wherein each dsRNA molecule comprises about 25 to about 30 base pairs, wherein each peptide comprise about 5 to about 40 aminoacid and comprise aminoacid sequence KVLKQ (SEQ ID NO:51), and wherein said dsRNA molecule is bonded to described peptide by yoke.In some embodiment, the aminoacid sequence of peptide can be to be selected from the group of being made up of following sequence: KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ IDNO:33);

KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：42)；

VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：43)；

AQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：44)；

KDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：45)；

KKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：46)；

KRSRKESYSVYVYKVLKQ(SEQ ID NO：47)；

RKESYSVYVYKVLKQ(SEQ ID NO：41)；

SYSVYVYKVLKQ(SEQ ID NO：48)；VYVYKVLKQ(SEQ ID NO：49)；

YKVLKQ (SEQ ID NO:50); And KVLKQ (SEQ ID NO:51).

In other embodiment, dsRNA has 5 of 2 or more bp ' and dangles, perhaps 3 of 2 or more bp ' dangle, wherein this to dangle can be in sense strand and/or antisense strand one or two.In embodiment further, dsRNA does not dangle.In embodiment further, dsRNA has length and is about 25bp chain of about 29bp extremely.In embodiment further, the dsRNA molecule contains one adopted RNA chain and an antisense RNA chain, and a peptide is bonded to 5 of antisense strand ' end by yoke.

On the other hand, the invention provides compositions, comprise pharmaceutical composition, it is suitable for animal is used one or more siRNA molecules, wherein said composition comprises one or more siRNA molecules and one or more peptides, wherein each siRNA molecule comprises about 25 double-stranded ribonucleotides to about 30 base pairs, and wherein each peptide be about 5 to about 40 aminoacid and comprise aminoacid sequence KVLKQ (SEQ ID NO:51), and wherein said peptide is bonded to described dsRNA by yoke.In one embodiment, described peptide is bonded to the molecule that is attached to the animal inner cell by yoke.In another embodiment, described siRNA molecule comprises with the sequence that comes from part TNF-α gene order.

In other other embodiment, the invention provides the polypeptide conjugates of siRNA molecule and those siRNA, wherein said siRNA comprises three chains, is called A, B1 and B2 (A:B1B2) here, wherein B1 and B2 be complementary to A non-overlapped district and also with its formation base pair (bp).Therefore, for the siRNA molecule in these embodiments, be to be different from the formed double stranded region of annealing fully by B2 and A by B1 and the A formed double stranded region of annealing.The A:B1 duplex can be separated with the A:B2 duplex by " breach (gap) ", described " breach " results from least one unpaired nucleotide that is located between A:B1 duplex and the A:B2 duplex in the A chain, and is different from any one or a plurality of nucleotide in pairs on 3 ' end of A, B1 and/or B2 chain one of them or two fully.Perhaps, the A:B1 duplex can be separated with the A:B2 duplex by " otch (nick) ", in the A chain, be not located in the unpaired nucleotide between A:B1 duplex and the A:B2 duplex like this, make unique unpaired nucleotide, if any, be on 3 ' end of A, B1 and/or B2 chain one of them or two.

Typically, the siRNA of these aspects comprises that about 15 base pairs of total are to about 40 base pairs according to the present invention; More typically, about 18 to about 35 base pairs; Further about more typically 20 to 30 base pairs; 21,22,23,24,25,26,27,28 or 29 base pairs the most typically.Described siRNA can randomly comprise the strand 3 of 1 nucleotide to 5 nucleotide ' dangle.The most typically, this strand 3 ' dangle is 1,2,3 or 4 nucleotide.

Therefore, this three chain siRNA of the present invention comprise an A sense strand or an A antisense strand, and the length of wherein said A chain is that about 15 nucleotide are to about 50 nucleotide; The length of described more typically A chain is that about 18 nucleotide are to about 40 nucleotide; Further more typically, the length of described A chain is that about 20 nucleotide are to about 32 nucleotide; The most described A chain length is 21,22,23,24,25,26,27,28,29,30 or 31 nucleotide.

In addition, three chain siRNA of the present invention comprise two or more B chains, are called here, for example, B1 and B2, wherein each B chain is complementary to the non-overlapped district of homology A chain, and wherein a B chain (B1) is to separate with the breach and the 2nd B chain (B2) of an otch or one or more nucleotide.Whether be sense strand or antisense strand, each B chain will be antisense strand or sense strand respectively if depending on homology A chain.Each B chain described here (B1, B2, etc.) length independently of one another is that about 1 nucleotide is to about 25 nucleotide; More typically, length is that about 4 nucleotide are to about 20 nucleotide; Further length is that about 5 nucleotide are to about 16 nucleotide more typically; Length is 6,7,8,9,10,11,12,13,14 or 15 nucleotide the most typically.

Depend on desired definite application, a B chain (B1) can be separated with otch or with breach and the 2nd B chain (B2).In those embodiments, wherein B1 and B2 separate with breach, and this breach is that about 1 nucleotide is to about 25 nucleotide typically; This breach is that about 1 nucleotide is to about 15 nucleotide more typically; Further this breach is that about 1 nucleotide is to about 10 nucleotide more typically; This breach is 1,2,3,4,5,6,7,8 or 9 nucleotide the most typically.Each B chain can, independently of one another, with 5 ' hydroxyl (that is, 5 '-OH) stop or can with 5 ' phosphoric acid (promptly 5 '-PO ₄) stop.

In other respects, the invention provides the double-stranded siRNA molecule that utilizes one or more jagged or otch and comprise the method for compositions of the double-stranded siRNA molecule of one or more jagged or otch.

In some embodiment, method described here comprises the following steps, (a) chooses target gene, and wherein said target gene is the target viral gene, is used for the gene silencing that siRNA mediates; (b) design and/or synthetic suitable siRNA molecule, the gene silencing that siRNA mediated that is used for the target viral gene, wherein said siRNA molecule includes the duplex of breach or otch, and wherein said breach or otch appears at or sense strand in or in antisense strand; (c) described siRNA is applied to the cell of expressing the target viral gene, wherein said siRNA can be bonded to corresponding target virus mRNA specifically, thereby reduces its expression in cell.

In selective embodiment, method disclosed herein comprises following step, (a) choose the target gene that is used for siRNA institute mediated gene silencing, wherein said target gene be endogenous gene and, randomly, wherein said endogenous target gene comprises that one or more are different from the sequence variations of corresponding wild type endogenous gene; (b) the double-stranded siRNA molecule of design and/or synthetic suit jagged or otch, the gene silencing that siRNA mediated that is used for the endogenous target gene, wherein said siRNA molecule includes the duplex of breach or otch, and wherein said breach or otch appears at or sense strand in or in antisense strand; (c) described siRNA molecule is applied to the cell of expressing the endogenous target gene, wherein said siRNA can be bonded to corresponding endogenous said target mrna specifically, thereby reduces its expression in cell.

Should be understood that method of the present invention does not need the early stage nucleotide sequence of understanding by jagged or otch double-stranded siRNA targeting, each possibility gene mutation.At first, the nucleotide sequence of siRNA can be the conserved region that is selected from target gene.

Compositions disclosed herein and method are being useful aspect the various target virus titers of minimizing, and described target virus includes, but are not limited to, retrovirus, such as human immunodeficiency virus (HIV), and Respirovirus, such as the human respiratory syncytial virus, human metapneumovirus, the human parainfluenza viruses 1, the human parainfluenza viruses 2, and the human parainfluenza viruses 3, human parainfluenza viruses 4a, human parainfluenza viruses 4b, influenza A virus, Influenza B virus, rhinovirus and influenza virus C.

On the other hand, the invention provides the method that a kind of suppressor gene is expressed in animal, it comprises uses the compositions that comprises one or more double stranded RNAs (dsRNA) molecule to animal, said composition comprises pharmaceutical composition, wherein said pharmaceutical composition comprises described dsRNA molecule and peptide, wherein each dsRNA molecule be about 25 to about 30 base pairs, wherein each peptide be about 5 to about 40 aminoacid and, typically, comprise aminoacid sequence KVLKQ (SEQ ID NO:51) and wherein each dsRNA molecule be bonded on the peptide by yoke.

The summary of accompanying drawing and sequence identifier

Fig. 1: have polypeptide PN277 and be bonded to the dsCoP277nfR952 of siRNA N163 and the gel electrophoresis of the external dicer digestion product of dsCoP277nfR950 conjugates by yoke.

Fig. 2: RP-HPLC analyzes dicer Cobra venom endonuclease processing dynamics that non-yoke closes the siRNAN163 duplex.(A) unprocessed N163 duplex is (B) with dicer Cobra venom endonuclease incubation 1 hour, (C) with dicer Cobra venom endonuclease incubation 2.5 hours, (D) with dicer Cobra venom endonuclease incubation 5 hours with (E) with dicer Cobra venom endonuclease incubation 7 hours.

Fig. 3: non-yoke shown in the RP-HPLC analysis chart 2 closes the dicer Cobra venom endonuclease processing dynamics of siRNA N163 duplex and learns chart.

Fig. 4: ESI-MS analyzes the dicer digestion in 7 hours that non-yoke closes N163.

Fig. 5: ESI-MS analysis has polypeptide PN857 and is bonded to dicer Cobra venom endonuclease processing siRNA N163, that yoke closes siRNA by yoke.(A) 8 hours blanks that do not have that the dicer Cobra venom endonuclease exists and (B) 8 hours dicer endonuclease enzymic digestion conjugates.

SEQ ID NO:1 is aminoacid sequence KRRQRRR, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:2 is aminoacid sequence RQIKIWFQNRRMKWKK, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:3 is an aminoacid sequence

DAATATRGRSAASRPTERPRAPARSASRPRRPVD, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:4 is aminoacid sequence AAVALLPAVLLALLAP, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:5 is aminoacid sequence AAVLLPVLLPVLLAAP, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:6 is aminoacid sequence VTVLALGALAGVGVG, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:7 is aminoacid sequence GALFLGWLGAAGSTMGA, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:8 is aminoacid sequence MGLGLHLLVLAAALQGA, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:9 is aminoacid sequence LGTYTQDFNKFHTFPQTAIGVGAP, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:10 is aminoacid sequence GWTLNSAGYLLKINLKALAALAKKIL, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:11 is aminoacid sequence TPPKKKRKVEDPKKKK, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:12 is aminoacid sequence RRRRRRR, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:13 is aminoacid sequence KLALKLALKALKAALKLA, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:14 is aminoacid sequence GLFGAIAGFIENGWEG, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:15 is aminoacid sequence FFGAVIGTIALGVATA, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:16 is aminoacid sequence FLGFLLGVGSAIASGV, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:17 is aminoacid sequence GVFVLGFLGFLATAGS, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:18 is aminoacid sequence GAAIGLAWIPYFGPAA, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:19 is an aminoacid sequence

ACTCPYCKDSEGRGSGDPGKKKQHICHIQGCGKVYGKTSHLRAHLRWHTGERPFMC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:20 is an aminoacid sequence

ACTCPNCKDGEKRSGEQGKKKHVCHIPDCGKTFRKTSLLRAHVRLHTGERPFVC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:21 is an aminoacid sequence

ACTCPNCKEGGGRGTNLGKKKQHICHIPGCGKVYGKTSHLRAHLRWHSGERPFVC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:22 is an aminoacid sequence

ACSCPNCREGEGRGSNEPGKKKQHICHIEGCGKVYGKTSHLRAHLRWHTGERPFIC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:23 is an aminoacid sequence

RCTCPNCTNEMSGLPPIVGPDERGRKQHICHIPGCERLYGKASHLKTHLRWHTGER PFLC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:24 is an aminoacid sequence

PCDCPNCQEAERLGPAGVHLRKKNIHSCHIPGCGKVYGKTSHLKAHLRWHTGERPF VC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:25 is an aminoacid sequence

RCTCPNCKAIKHGDRGSQHTHLCSVPGCGKTYKKTSHRRAHLRKHTGDRPFVC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:26 is an aminoacid sequence

PQISLKKKIFFFIFSNFRGDGKSRIHICHLCNKTYGKTSHLRAHLRGHAGNKPFAC, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:27 is an aminoacid sequence

WWETWKPFQCRICMRNFSTRQARRNHRRRHR, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:28 is aminoacid sequence GKINLKALAALAKKIL, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:29 is aminoacid sequence RVIRVWFQNKRCKDKK, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:30 is an aminoacid sequence

GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:31 is aminoacid sequence GEQIAQLIAGYIDIILKKKKSK, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:33 is an aminoacid sequence

KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:35 is a nucleotide sequence

5 '-AUGGUGUGGGUGAGGAGCACAUGGGUG-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:36 is a nucleotide sequence

5 '-CCCAUGUGCUCCUCACCCACACCdAT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:37 is an aminoacid sequence

KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:41 is aminoacid sequence RKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:42 is an aminoacid sequence

KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:43 is an aminoacid sequence

VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:44 is an aminoacid sequence

AQKKDGKKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:45 is aminoacid sequence KKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:46 is aminoacid sequence KKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:47 is aminoacid sequence KRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:48 is aminoacid sequence SYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:49 is aminoacid sequence VYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:50 is aminoacid sequence YKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:51 is aminoacid sequence KVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:52 is an aminoacid sequence

KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ, an exemplary peptides that is suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:53 be nucleotide sequence 5 '-GCCUGUACCUCAUCUACUCUU-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:54 is a nucleotide sequence

3 '-UUCGGACAUGGAGUAGAUGAG-5 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:55 is a nucleotide sequence

5 '-GCCUCUUCUCCUUCCUGAUCGUGdGdC-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:56 is a nucleotide sequence

3 '-GUCGGAGAAGAGGAAGGACUAGCACCG-5 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:57 is a nucleotide sequence

5 '-GCCUGCUGCACUUUGGAGUGAUCdGdG-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:58 is a nucleotide sequence

3 '-GACGGACGACGUGAAACCUCACUAGCC-5 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:59 is a nucleotide sequence

5 '-CCCAUGUGCUCCUCACCCACACCdAT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:60 is a nucleotide sequence

3 '-GUGGGUACACGAGGAGUGGGUGUGGUA-5 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:61 is a nucleotide sequence

5 '-ACCUCAUCUACUCCCAGGUCCUCdTdT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:62 is a nucleotide sequence

3 '-CAUGGAGUAGAUGAGGGUCCAGGAGAA-5 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:63 is a nucleotide sequence

5 '-GCCUGUACCUCAUCUACUCCCAGGUCC-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:64 is a nucleotide sequence

5 '-GGUCCUGGGAGUAGAUGAGGUACAGGCUU-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:65 is a nucleotide sequence

5 '-AGACAGCGACCAAAAGAAUUCGGdAdU-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:66 is a nucleotide sequence

5 '-AUCCGAAUUCUUUUGGUCGCUGUCUdTdT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:67 is a nucleotide sequence

5 '-AUGAAGAUCUGUUCCACCAUUGAdAdG-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:68 is a nucleotide sequence

5 '-CUUCAAUGGUGGAACAGAUCUUCAUdTdT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:69 is a nucleotide sequence

5 '-GAUCUGUUCCACCAUUGAAGAACdUdC-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:70 is a nucleotide sequence

5 '-GAGUUCUUCAAUGGUGGAACAGAUCdTdT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:71 is a nucleotide sequence

5 '-UUGAGGAGUGCUGAUUAAUGAUdCdC-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:72 is a nucleotide sequence

5 '-GGAUCAUUAAUCAGGCACUCCUCAAdTdT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:73 is a nucleotide sequence

5 '-GGAUCUUAUUUCUUCGGAGACAAdTdG-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:74 is a nucleotide sequence

5 '-CAUUGUCUCCGAAGAAAUAAGAUCCdTdT-3 ', exemplary polynucleotide that are suitable for producing as siRNA-peptide conjugates described here.

SEQ ID NO:75 is an exemplary 25-mer B chain-ordering

5 '-nucleotide sequence of GGAUCUUAUUUCUUCGGAGACAAUG-3 ', be suitable for producing as table 2 here described in siRNA-peptide conjugates.

SEQ ID NO:76 is an exemplary 27-mer A chain-ordering

3 '-nucleotide sequence of TTCCUAGAAUAAAGAAGCCUCUGUUAC-5 ', be suitable for producing as table 2 here described in siRNA-peptide conjugates.

SEQ ID NO:77-132 is A, B1 and the B2 chain nucleotide sequence of exemplary polynucleotide, is used for producing siRNA-peptide conjugates jaggy described in table 2 here.

SEQ ID NO:34,38-40,133 and 166 are A, B1 and B2 chain nucleotide sequences of exemplary polynucleotide, are used to produce the siRNA-peptide conjugates that otch is arranged as described here.

The detailed description of invention

The present invention is based on such observation, short interfering nucleic acid (siNA) or its precursor, unite use with the polypeptide that improves delivery of polynucleotides, for example show the sending property of improving, the stability of raising and/or the toxicity that reduces during the people when being applied to mammal in the body. Therefore, the polypeptide of raising delivery of polynucleotides disclosed herein can be natural polypeptides or artificial polypeptide, and they are selected, mainly is to send or absorb because they can improve in the cell that polypeptide comprises siNA and their precursors.

SiNA of the present invention and composition can be mixed together or be compound in or yoke is bonded to the polypeptide that one or more improve delivery of polynucleotides, improve the composition of sending in the siNA cell in order to form, than the sending of obtaining with naked siNA (that is, not sending the siNA that improves the polypeptide existence) contact target cell.

Improve the polypeptide Choice and design of delivery of polynucleotides

In some embodiment of the present invention, the polypeptide that improves delivery of polynucleotides is histone, perhaps its polypeptide or fragments of peptides, derivative, analog, perhaps its conjugates. In these embodiments, siNA be with one or more total length histones or at least in part corresponding to the polypeptide of histone partial sequence be mixed together, compound or yoke closes, one or more following histones for example: histone h1, histone H2A, histone H2B, histone H 3 or histone H 4, perhaps their one or more polypeptide fragments or derivative, this fragment or derivative comprise at least part of sequence of histone, typically the continuous residue of 5-10 or 10-20 natural (native) histone at least.

In more detailed embodiment, siRNA/ histone mixture, compound or conjugates are substantially free of amphiphilic compound. In other detailed embodiments, mix with histone or polypeptide, siNA compound or that yoke closes comprises double-stranded RNA, for example have 30 or Oligonucleotide and be the double-stranded RNA of short interfering rna (siRNA) more. In exemplary, the polypeptide that histone improves delivery of polynucleotides comprises histone H2B, as following polypeptide (being called PN73) described, that improve delivery of polynucleotides is exemplified here. In other detailed embodiment, the polypeptide that improves delivery of polynucleotides can be Pegylation, in order to improve stability and/or validity, uses in the situation especially in vivo.

In other embodiment of the present invention, the polypeptide of the raising delivery of polynucleotides of selected or appropriate design comprises amphipathic amino acid sequence. For example, selected polypeptide useful, that improve delivery of polynucleotides comprises a plurality of nonpolar or hydrophobic amino acid residues that forms hydrophobicity sequence territory or motif, they are connected to a plurality of charge residue residues that form charged sequence territory or motif, obtain peptide amphiphile.

In other embodiment, polypeptide selected, that improve delivery of polynucleotides comprises protein transduction domain or motif, and fusogenic peptide territory or motif. Protein transduction domain is the peptide sequence that can insert and preferably pass through (transit through) cell membrane. The peptide that fusogenic peptide is stabilization removal (destablized) lipid film (for example, plasma membrane or around the film of endosome), can be enhanced under low pH. Exemplary fused peptide territory or motif are found in the diversified virus amalgamation protein and at other albumen for example in the fibroblast growth factor 4 (FGF-4).

For reasonable design the present invention improves the polypeptide of delivery of polynucleotides, protein transduction domain is used as helping nucleic acid to enter the motif of cell by plasma membrane. In some embodiment, the nucleic acid of being transported can be encapsulated in the endosome. The inside of endosome has low pH value, causes fusogenic peptide motif stabilization removal endosome film. The stabilization removal of endosome film and breaking so that siNA is released in the kytoplasm, siNA can with the associating of RISC compound, and be oriented to its said target mrna.

The protein transduction domain example is used for choosing wantonly and mixes the polypeptide that the present invention improves delivery of polynucleotides, comprising:

1.TAT protein transduction domain (PTD) (SEQ ID NO:1) KRRQRRR;

2. wear film peptide PTD (SEQ ID NO:2) RQIKIWFWNRRMKWKK;

3.VP22 PTD(SEQ ID NO：3)

DAATATRGRSAASRPTERPRAPARSASRPRRPVD；

4. card Podbielniak (Kaposi) FGF burst (SEQ ID NO:4) AAVALLPAVLLALLAP, and (SEQ ID NO:5) AAVLLPVLLPVLLAAP;

5. people β 3 integrin signaling sequences (SEQ ID NO:6) VTVLALGALAGVGVG;

6.gp41 fusion sequence (SEQ ID NO:7) GALFLGWLGAAGSTMGA;

7. caimans (Caiman crocodylus) Ig (v) light chain (SEQ ID NO:8) MGLGLHLLVLAAALQGA;

8.hCT-derived peptide (SEQ ID NO:9) LGTYTQDFNKFHTFPQTAIGVGAP;

9. transporter (Transportan) (SEQ ID NO:10) GWTLNSAGYLLKINLKALAALAKKIL;

10.Loligomer(SEQ ID NO：11)TPPKKKRKVEDPKKKK；

11. arginine peptide (SEQ ID NO:12) RRRRRRR; With

12. amphipathic model peptide (SEQ ID NO:13) KLALKLALKALKAALKLA.

The example of virus fusion peptide fusion area is used for choosing wantonly and mixes the polypeptide that the present invention improves delivery of polynucleotides, comprising:

1. influenza HA 2 (SEQ ID NO:14) GLFGAIAGFIENGWEG;

2. celestial platform F1 (SEQ ID NO:15) FFGAVIGTIALGVATA;

3. respiratory syncystial virus F 1 (SEQ ID NO:16) FLGFLLGVGSAIASGV;

4.HIV gp41 (SEQ ID NO:17) GVFVLGFLGFLATAGS; With

5. Ebola GP2 (SEQ ID NO:18) GAAIGLAWIPYFGPAA.

In other embodiment of the present invention, the invention provides and improve the polypeptide that polynucleotide are sent, they mix DNA in conjunction with territory or motif, help the formation of polypeptide-siNA complex and/or improve sending of siNA in method and composition of the present invention.Exemplary DNA comprises various " zinc refers to " territory in conjunction with the territory in this context, as be used for DNA in conjunction with modulability albumen and other albumen of being differentiated in the following Table 1 described (referring to, for example, Simpson etc., J.Biol.Chem.278:28011-28018,2003).

Table 1

The protein-bonded exemplary zinc-finger motif of different DNA

* this table has illustrated the bonded conservative zinc-finger motif of double-stranded DNA, be characterized by C-x (2,4)-and C-x (12)-H-x (3)-H (SEQ ID NO:32) motif pattern, according to the present invention, itself can be used to select and design the polypeptide that other raising polynucleotide are sent.

Sequence Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5 and Y4pB1A.4 shown in the * table 1 are denoted as SEQ ID NO:19,20,21,22,23,24,25 and 26 here.

Improve that useful selected DNA comprises in conjunction with the territory aspect the polypeptide that polynucleotide send making up the present invention, for example, the part of HIV Tat protein sequence (seeing the following examples).

Below in these described illustrative embodiments of the invention, mediation has improved the single polypeptide that the siNA that sends enters target cell by any aforesaid structural detail, territory or motif being combined as effectively, can reasonably design and make up the polypeptide that the raising polynucleotide are sent.For example, the protein transduction domain of TAT polypeptide is merged terminal 20 aminoacid to the proteic N-of the influenza virus hemagglutinin that is known as HA2, to produce the polypeptide that exemplary raising polynucleotide are sent.Provide in the present invention various other, improve the polypeptide construct that polynucleotide are sent, proved that notion of the present invention is widely applicable for the polypeptide of creating and using effective raising polynucleotide of various assembling to send, and is used to improve siNA and sends.

The polypeptide that other in the present invention exemplary raising polynucleotide are sent can be to be selected from following peptide: WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO:27); GKINLKALAALAKKIL (SEQ ID NO:28), RVIRVWFQNKRCKDKK (SEQID NO:29),

GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO:30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO:31), poly-Lys-Trp, 4:1, MW20,000-50,000; With poly-Orn-Trp, 4:1, MW 20,000-50,000.The peptide that useful, other raising polynucleotide are sent in compositions here and the method comprises melittin (mellitin) protein sequence whole or part.

The polypeptide that other exemplary raising polynucleotide are sent is specified in the following example.Any one of these peptides or combination can be select or combination effectively improve the polypeptide reagent that polynucleotide are sent so that produce, send in the cell of siNA in the inventive method and the compositions to induce or to help.

The compositions that comprises polypeptide, siRNA and lipid that the raising polynucleotide are sent

Aspect more detailed, comprising one or more siRNA and improve mixture, complex and/or the conjugates of the polypeptide that polynucleotide send can be randomly and cation lipid such as LIPOFECTIN

Combination (for example mixing or compound).In this context, against expectation disclosed at this, only be bonded to the polypeptide that the raising polynucleotide of siRNA send and realized sending of siNA by compound or yoke, be enough to use the RANi mediated gene silencing.Disclosed further at this, siRNA/ improves polypeptide complex or the conjugates that polynucleotide are sent, and when mixing or compound tense with cation lipid such as lipofectin, demonstration mediation siNA sends the bigger activity with gene silencing.

In order to produce these compositionss of the polypeptide, siRNA and the cation lipid that comprise that the raising polynucleotide are sent, at first siRNA and peptide can be mixed together in the suitable media such as cell culture medium, add cation lipid to the mixture that obtains then, send peptide/cation lipid compositions to form siRNA/.Randomly, at first peptide and cation lipid are mixed together in the suitable media such as cell culture medium, add siRNA subsequently and send peptide/cation lipid compositions to form siRNA/.

Of the present invention aspect these, useful cation lipid example comprises N-[1-(2,3-two oily acyloxy) propyl group]-N, N, the N-trimethyl ammonium chloride, 1, two (oily acyloxy)-3-3-(trimethyl ammonium) propane of 2-, 1,2-two Semen Myristicae oxygen base propyl group-3-dimethyl ethoxy ammonium bromide, and GERBU Adjuvant 100,2, two oily acyloxy-N-[2 (spermine formamido) ethyls of 3-]-N, N-dimethyl-1-propyl group trifluoroacetic acid ammonium salt, 1,3-two oily acyloxy-2-(6-carboxyl spermine base)-propyl amides, the two octadecyl amide of 5-carboxyl spermine base glycine, tetramethyl four palmityl spermine, tetramethyl four oleyl spermine, tetramethyl osmanthus in April alkyl spermine, tetramethyl four myristyl spermine and tetramethyl two oleyl spermine.(N-[1-(2 for DOTMA, 3-two oily acyloxy) propyl group]-N, N, the N-trimethyl ammonium chloride), DOTAP (1, two (the oily acyloxy)-3 of 2-, 3-(trimethyl ammonium) propane), DMRIE (1,2-two Semen Myristicae oxygen base propyl group-3-dimethyl-ethoxy ammonium bromide) or DDAB (GERBU Adjuvant 100).The polyvalent cation lipid comprises fat spermine class (lipospermine), more particularly, DOSPA (2,3-two oily alkene oxygen base-N-[2 (spermine formamido) ethyls]-N, N-dimethyl-1-propyl group trifluoroacetic acid ammonium salt) and DOSPER (1,3-two oily acyloxy-2-(6 carboxyl spermine base)-propyl group-amide, and two-and four-alkyl-four-methyl spermine class, include but not limited to TMTPS (tetramethyl four palmityl spermine), TMTOS (tetramethyl four oleyl spermine), TMTLS (tetramethyl osmanthus in April alkyl spermine), TMTMS (tetramethyl four myristyl spermine) and TMDOS (tetramethyl two oleyl spermine), DOGS (two octadecyls-amide groups glycyl spermine (TRANSFECTAM ).Other useful cation lipid is described in, for example, and U.S. Patent number 6,733,777; U.S. Patent number 6,376,248; U.S. Patent number 5,736,392; U.S. Patent number 5,686,958; U.S. Patent number 5,334,761 and U.S. Patent number 5,459,127.

Cation lipid is right and wrong cation lipid, particularly neutral lipid randomly, combines, and this class neutral lipid is lipid such as DOPE (DOPE) for example, DPhPE (two phytane acyl PHOSPHATIDYL ETHANOLAMINE) or cholesterol.Consist of 3: 1 (w/w) mixture or DOTMA and the DOPE (LIPOFECTIN of DOSPA and DOPE

, the cation lipid compositions of 1: 1 (w/w) mixture Invitrogen) is useful in transfection group compound of the present invention usually.Preferred transfection group compound is meant those of inducing the higher eukaryotic cell lines of transfection basically.

The selection of short interfering nucleic acid (siNA), design and synthetic

In exemplary embodiment, of the present disclosure being characterized as comprises the small nucleic acids molecule, such as short interfering nucleic acid (siNA), short interfering rna (siRNA), double-stranded RNA (dsRNA), microRNA (mRNA), the perhaps compositions of short hairpin RNA (shRNA), described small nucleic acids molecular mixing or be compound in, perhaps yoke is bonded to and improves the polypeptide that polynucleotide are sent.

As used herein, term " short interfering nucleic acid ", " siNA ", " short interfering rna ", " siRNA ", " short interfering nucleic acid molecule ", " the short oligonucleotide molecules of disturbing " or " the short interfering nucleic acid molecule of chemical modification ", be meant arbitrary nucleic acid molecules, it can inhibition or down-regulation of gene expression or virus replication, for example, rely on the interference of sequence-specific mode mediate rna " RNAi " or gene silencing.In exemplary, siNA is a kind of double-stranded polynucleotide molecule, comprise self-complementary have justice and antisense district, wherein said antisense district comprises and is complementary to nucleotide sequence or its a part of nucleotide sequence that is used to reduce expression in the target nucleic acid molecule, and describedly have justice district to comprise nucleotide sequence corresponding to (that is, it is to be equal to basically aspect the sequence) target nucleic acid sequence or its part.

" siNA " represents small RNA, siRNA for example, i.e. and the double-strandednucleic acid of short length (perhaps randomly its longer precursor), and be not unacceptable toxicity in target cell.In the present invention, useful siNA length is optimised for about length of 21 to 23bp in some embodiment.However, comprise that at useful siNA there is no particular limitation aspect the siRNA length.For example, siNA can be presented (presented) to cell at first with the form of precursor, promptly is different in essence in the finally or form processing of siNA, and will there be and brings into play the activity of gene silencing in it after being delivered to target cell or sending.The precursor forms of siNA for example, can comprise the precursor sequence element, they are processed, be degraded, be changed or be cut in or time of sending after, thereby be active siNA mediated gene silencing so that be created in the cell.Therefore, in some embodiment, the forebody length that useful in the present invention siNA has is, for example, about 100-200 base pair, 50-100 base pair, perhaps less than about 50 base pairs, it can produce siNA active, that processed in target cell.In other embodiment, useful siNA or siNA forebody length are approximately 10 to 49bp, and 15 to 35bp, or about 21 to 30bp.

Disclosed immediately exemplary siNA molecule is chemosynthesis.Oligonucleotide is to use scheme known in the art and synthetic, for example, as is described in Caruthers etc., (method in the zymetology) Methods in Enzymology 211:3-19,1992; Thompson etc., International PCT publication No. WO99/54459; Wincott etc., Nucleic Acids Res.23:2677-2684,1995; Wincott etc., Methods Mol.Bio.74:59,1997; Brennan etc., Biotechnol.Bioeng.61:33-45,1998; And Brennan, U.S. Patent number 6,001,311.But the synthetic of siNA molecule of the present invention's chemical modification comprises: (a) the siNA molecule of synthetic two complementary strands; (b) be fit to obtain under the condition of double-stranded siNA molecule these two complementary strands of annealing together.In another embodiment, the synthetic of two complementary strand siNA molecules is synthetic by the solid phase oligonucleotide.In another embodiment, the synthetic of two complementary strand siNA molecules is synthetic by solid phase series connection oligonucleotide.

Oligonucleotide (for example, some modified oligonucleotide or lack the oligonucleotide part of ribonucleotide) be that to use scheme as known in the art synthetic, for example, as be described in Caruthers etc., (method in the zymetology) Methods in Enzymology 211:3-19,1992; Thompson etc., International PCT publication No. WO 99/54459; Wincott etc., Nucleic Acids Res.23:2677-2684,1995; Wincott etc., Methods Mol.Bio.74:59,1997; Brennan etc., BiotechnolBioeng.61:33-45,1998; And Brennan, U.S. Patent number 6,001,311.RNA comprises the synthetic of some siNA molecule of the present invention, follows method in common, for example, as is described in Usman etc., J.Am.Chem.Soc.109:7845,1987; Scaringe etc., Nucleic Acids Res.18:5433,1990; With Wincott etc., Nucleic Acids Res.23:2677-2684,1995; Wincott etc., Methods Mol.Bio.74:59,1997.

In some embodiment of the present invention,, use and improve the polypeptide that polynucleotide are sent, to help to send the nucleic acid precursor that the nucleic acid molecules bigger than conventional siNA comprises that siNA is big as top institute note.For example, on behalf of the more large nucleic acids of " precursor ", the method and composition here can be used to improve be delivered to desired siNA, wherein said precursor aminoacid, before being delivered to target cell, in the process or afterwards, can be cut or otherwise processing, so that form active siNA, be used for expressing in the target cell regulator gene.For example, siNA precursor polynucleotide can be chosen as the polynucleotide of annular, strand, have two or more circuluses and one and comprise the complementary stem that justice and antisense district are arranged of oneself, wherein said antisense district comprises and is complementary to nucleotide sequence or its a part of nucleotide sequence in the target nucleic acid molecule, and describedly have justice district to have corresponding to target nucleic acid sequence or its a part of nucleotide sequence, with wherein said annular polynucleotide can be in vivo or external processed so that produce can mediate rna i active siNA molecule.

In mammalian cell, the dsRNA that is longer than 30 base pairs can activate dsRNA dependent kinases PKR and 2 '-5 '-oligoadenylate synthetase, and this is normally by interferon-induced.Activated PKR and suppressed general translation with phosphorylation translation factor eukaryotic initiation factor 2 α (eIF2 α), and 2 '-5 '-oligoadenylate synthetase causes the degraded of non-specific mRNA by the activation of RNase L.By means of their small size (being meant non-precursor forms especially), normally less than 30 base pairs and the most frequently used about 17-19,19-21 or 21-23 base pair, siNA of the present invention has avoided the activation of interferon response.

Than the nonspecific action of long dsRNA, siRNA can mediate the selected gene silence in mammlian system.Hairpin RNA has the ring of a weak point and have 19 to 27 base pairs, the equally optionally expression of silent gene, the sequence of described dna homolog in double-stranded stem in stem.Mammal can be converted into siRNA with short hairpin RNA, so that mediation selected gene silence.

The RISC mediation has the single stranded RNA cutting of the sequence that is complementary to siRNA duplex antisense strand.The cutting of target RNA occurs in the middle part, zone that is complementary to siRNA duplex antisense strand.Research show, the siRNA duplex of 21 nucleotide when comprise two nucleotide 3 '-be the most active when dangling.In addition, with 2 '-deoxidation (2 '-H) or 2 '-the O-methyl nucleotide replaces the activity that one or two siRNA chain has reduced RNAi in fact fully, yet existing report, with Deoxydization nucleotide (2 '-H) replacement 3 '-the terminal siRNA nucleotide that dangles can tolerate.

Research shows, replace with deoxyribonucleotide to have 2 nucleotide 3 ' the 21-mersiRNA duplex 3 that dangles '-fragment of dangling, the RNAi activity there is not adverse influence.Existing report reaches 4 nucleotide with the deoxyribonucleotide replacement and well can tolerate on each end of siRNA, cause not having the RNAi activity yet replace with deoxyribonucleotide fully.

In some embodiments of the present invention, dsRNA has 5 of 2 or a plurality of bp ' and dangles, perhaps 3 of 2 or a plurality of bp ' to dangle, described here dangling can be to have on justice or the antisense strand.In some embodiments, dsRNA does not dangle.In some embodiments, to have length be 27bp to 29 bp to dsRNA.In some embodiments, the dsRNA molecule comprises one adopted RNA chain and an antisense RNA chain, and peptide is bonded to 5 of antisense strand ' end by yoke.

Perhaps, siNA can be sent, as by the single or multiple siNA of coding and be oriented to the expressed single or multiple transcription products of polynucleotide carrier that their are expressed in target cell.In these embodiments, wait that in target cell the double-stranded of the final transcription product of siRNA of being expressed partly can be, for example, 15 to 49 bp, 15 to 35 bp, the length of perhaps about 21 to 30 bp.In exemplary embodiment, the double-stranded part of siNA, here two strands be made into right, be not limited to complete paired nucleotide fragments, and can comprise not pairing, projection (bulge) (on a chain, lacking corresponding complementary nucleotide), dangle etc. because of mispairing (corresponding nucleotide be not complementary).Can comprising not, the degree of mating section is the formation that they do not disturb siNA.In more detailed embodiment, " projection " can comprise 1 to 2 unpaired nucleotide, and its double center chain is made into right siNA double stranded region and can comprises about 1 to 7, perhaps about 1 to 5 projection.In addition, the quantity that " mispairing " part that is comprised in the siNA double stranded region can exist is about 1 to 7 or about 1 to 5.Modal mispairing situation, one of them nucleotide are guanines and another is a uracil.This mispairing is attributable to, and for example, has in the corresponding DNA of adopted RNA from C to T at coding, G is to A or their blended sudden changes, and other cause has also been considered in addition.In addition, in the present invention, its double center chain be made into right siNA double stranded region can be included in specific about quantitative range projection and by two kinds of mispairing parts.

The end structure of siNA of the present invention can be or (dangling) tack or viscosity, as long as this siNA keeps its reticent activity of expressing the target cell gene.(dangling) end structure of viscosity be not limited only to report as others 3 ' dangle.It antithesis, also can comprise 5 ' overhung structure, as long as can the reticent effect of induced gene for example use RNAi.In addition, the quantity of the nucleotide that dangles is not limited to report 2 or 3 nucleotide of limit, can also be quantity arbitrarily, as long as this dangles and does not damage the active for gene silencing of siNA.For example, dangling to comprise about 1 to 8 nucleotide, more commonly about 2 to 4 nucleotide.

Length with siNA of viscosity (dangling) end structure can be used on each end the duplex of pairing part and arbitrarily overhang express.For example one has the 25/27-mer siNA duplex that 2-bp 3 ' antisense dangles and has a 25-mer sense strand and a 27-mer antisense strand, and wherein paired part has 25bp length.

In addition, because the sequence of dangling can have the lower specificity to target gene, so it needn't complementary (antisense) or is equal to (justice is arranged) in target-gene sequence.In addition, as long as siNA can keep the effect of its gene silencing on target gene, it can comprise low-molecular-weight structure (for example natural RNA molecule such as tRNA, rRNA or viral RNA, perhaps engineered rna molecule), for example, and in the overhang at one end.

In addition, the end structure of siNA can have a stem-ring structure, here double-strandednucleic acid wherein the end of a side be with connexon nucleic acid for example connexon RNA connect.The length of double stranded region (stem-loop section) can be, for example, 15 to 49bp, and common ground 15 to 35bp and more commonly about 21 is to 30bp.Perhaps double stranded region is promptly waited the final transcription product of the siNA that is expressed in target cell, and its length can be, for example, about 15 to 49bp, and 15 to 35bp, perhaps about length of 21 to 30bp.When utilizing the connexon fragment, having no particular limits aspect the connexon length, as long as it does not hinder the pairing of stem portion.For example, for the inhibition of recombinating between the stable pairing of stem portion and the dna encoding, the connexon part can have Herba Trifolii Pratentis formula tRNA structure.Perhaps hinder the paired length of stem portion even connexon has, possiblely be, for example make up the connexon part that comprises intron, it is cut to be processed as this intron of process of mature rna at precursor RNA like this, makes the stem portion pairing by this.As for stem-ring siRNA, each end of the RNA of band shape structure (head or tail) can not have low molecular weight RNA.As mentioned above, these lower molecular weights RNA can comprise natural RNA molecule, such as tRNA, rRNA or viral RNA, and perhaps engineered rna molecule.

SiNA also can comprise have be complementary to nucleotide sequence in the target nucleic acid molecule or its a part of nucleotide sequence the strand polynucleotide (for example, this siNA molecule does not require in the siNA molecule and exists corresponding to target nucleic acid sequence or its a part of nucleotide sequence), here these strand polynucleotide can comprise the terminal phosphate group further, such as 5 '-phosphoric acid (sees, Martinez etc. for example, Cell.110:563-574,2002, with Schwarz etc., Molecular Cell 10:537-568,2002), perhaps 5 ', 3 '-diphosphonic acid.

As used herein, term siNA molecule is not limited to only comprise the molecule of natural RNA or DNA, also comprises the nucleotide and the non-nucleotide of chemical modification.

In some embodiment, short interfering nucleic acid molecule of the present invention lacks and comprises 2 '-hydroxyl (2 '-OH) nucleotide.In some embodiment, short interfering nucleic acid do not require have mediate rna i, have 2 '-nucleotide of oh group, therefore short interfering nucleic acid molecule of the present invention does not randomly comprise any ribonucleotide (for example have 2 '-nucleotide of OH group).But, this not requiring exists ribonucleotide to support the siNA molecule of RNAi in the siNA molecule, can have a connected connexon or a plurality of connexon, perhaps other are connected or by group, the parts of being united, perhaps comprise one or more and have 2 '-nucleotide chain of OH group.Randomly, the siNA molecule can comprise ribonucleotide on about nucleotide position of 5,10,20,30,40 or 50%.

As used herein, term siNA implication is to be equal to other terms that are used to describe nucleic acid molecules, this nucleic acid molecules can mediate sequence-specific RNA i, for example short interfering rna (siRNA), double-stranded RNA (dsRNA), little-RNA (mRNA), short hairpin RNA (shRNA), short oligonucleotide, short interfering nucleic acid, short modified oligonucleotide, chemical modification siRNA, the PTGS RNA (ptgsRNA) of disturbing of disturbing reach other.

In other embodiment, siNA molecule used in this invention can comprise independent adopted and antisense sequences or the district of having, wherein said have justice covalently to be connected by nucleotide or non-nucleotide connexon molecule with the antisense district, or by ionic interaction, hydrogen bond, Van der Waals force interaction, hydrophobic interaction and/or sedimentation alternately, non-covalently connect.

" antisense RNA " is to have the RNA chain that is complementary to target gene mRNA sequence, and is considered to induce RNAi by being bonded to target gene mRNA." adopted RNA is arranged " and have the sequence that is complementary to antisense RNA, and be annealed to its complementary antisense RNA so that form siRNA.These antisenses and have the available RNA synthesizer of adopted RNA synthetic routinely.

As used herein, term " RNAi construct " is to be used for this description generic term all the time, comprises siRNA (siRNA), hairpin RNA and can be cut in vivo to form other RNA types of siRNA.The RNAi construct here also comprises expression vector (also being known as the RNAi expression vector), and described expression vector can cause the transcript that forms dsRNA or hairpin RNA in cell, and/or can the interior transcript that produces siRNA of body.Randomly, siRNA comprises strand or double-stranded siRNA.

The siHybrid molecule is a kind of double-strandednucleic acid that is similar to the siRNA function that has.Be different from double stranded rna molecule, siHybrid is made up of a RNA chain and a DNA chain.Preferably, described RNA chain is the chain that antisense strand promptly is bonded to said target mrna.The siHybrid that is produced by the hybridization of DNA and RNA chain have a complementary portion of having hybridized and preferably at least one 3 ' end dangles.

The siNA that is applied among the present invention can assemble from two independent oligonucleotide (chain is that sense strand is an antisense strand with another here), wherein said antisense and sense strand are that complementary (that is, each chain comprises the nucleotide sequence that is complementary to nucleotide sequence in another chain to the oneself; Form duplex or duplex structure such as antisense strand and sense strand, for example wherein double stranded region is about 19 base pairs).Antisense strand can comprise and is complementary to nucleotide sequence or its a part of nucleotide sequence in the target nucleic acid molecule, and sense strand can comprise nucleotide sequence or its a part of nucleotide sequence corresponding to target nucleic acid sequence.Perhaps, siNA can assemble from single oligonucleotide, and wherein the self-complementary sense of siNA and antisense district use based on nucleic acid or based on the connexon mode of non-nucleic acid and be connected.

In other embodiments, the siNA that is used for sending in the cell according to the inventive method and compositions can be the polynucleotide that have duplex, asymmetrical duplex, hair clip or asymmetric hair clip secondary structure, have self-complementary sense and antisense district, wherein said antisense district comprises the nucleotide sequence that is complementary to independent target nucleic acid molecule or its a part of nucleotide sequence, and has the justice district to comprise corresponding to target nucleic acid sequence or its a part of nucleotide sequence.

In further embodiment, the invention provides the siNA of three chains, and compositions, they are mixed or be compound in or yoke is bonded to one or more and improves the polypeptide that polynucleotide are sent.SiRNA molecule described here comprises three chains, A, B1 and B2 (A:B1B2), wherein B1 and B2 chain be complementary to homology A chain non-overlapped district and also with its formation base pair (bp), wherein the B1 chain is to separate with corresponding B2 chain with otch or with breach.SiRNA molecule described here can start RNA and disturb cascade, causes the expression of target messenger RNA (mRNA) to be modified.

SiRNA molecule described here comprises three or many chains such as, example, A, B1 and B2 (A:B1B2), wherein B1 and B2 chain be complementary to A non-overlapped district and also with its formation base pair (bp); Be to be different from and not to be overlapped in the formed double stranded region of annealing wherein by B2 and A by B1 and the A formed double stranded region of annealing; Wherein the A:B1 duplex is separated by one " breach " and A:B2 duplex, described breach is produced by at least one unpaired nucleotide in the A chain, it between A:B1 duplex and A:B2 duplex, and it be different from A, B1 and/or B2 chain wherein one or both 3 ' end on any one or more unpaired nucleotides.

Perhaps, the molecule of siRNA described in the present invention comprises three or many chains such as, example, A, B1 and B2 (A:B1B2), wherein B1 and B2 chain be complementary to A non-overlapped district and also with its formation base pair (bp); Be to be different from and not to be overlapped in the formed double stranded region of annealing wherein by B2 and A by B1 and the A formed double stranded region of annealing; Wherein the A:B1 duplex is separated by " otch " between an A:B1 duplex and the A:B2 duplex and A:B2 duplex.

In one embodiment, A:B1B2 comprises, amounts to, and about 14 base pairs are to about 40 base pairs.In this embodiment, A represents sense strand and B1B2 represents antisense strand.A length is 15-50 nucleotide, and B1 and B2 are 1-20 nucleotide independently of one another; And the pattern length of B1+B2 is that about 8 nucleotide are to about 40 nucleotide.

In another embodiment, A:B1B2 comprises, amounts to, and about 16 base pairs are to about 40 base pairs.In this embodiment, A represents sense strand and B1B2 represents antisense strand.A length is 20-40 nucleotide, and B1 and B2 are 1-15 nucleotide independently of one another; And the pattern length of B1+B2 is that about 10 nucleotide are to about 30 nucleotide.

In another embodiment, A:B1B2 comprises, amounts to, and about 14 base pairs are to about 40 base pairs.In this embodiment, A represents antisense strand and B1B2 represents sense strand.A length is 15-50 nucleotide, and B1 and B2 are 1-20 nucleotide independently of one another; And the pattern length of B1+B2 is that about 8 nucleotide are to about 40 nucleotide.

In another embodiment, A:B1B2 comprises, amounts to, and about 16 base pairs are to about 40 base pairs.In this embodiment, A represents antisense strand and B1B2 represents sense strand.A length is 20-40 nucleotide, and B1 and B2 are 1-15 nucleotide independently of one another; And the pattern length of B1+B2 is that about 10 nucleotide are to about 30 nucleotide.

As implied above, three chain siRNA disclosed herein comprise that typically about 15 base pairs of total are to about 40 base pairs; More typically, about 18 to 35 base pairs; About more typically 20 to 30 base pairs; 21,22,23,24,25,26,27,28 or 29 base pairs the most typically.In some embodiment, siRNA can randomly comprise the strand 3 of 1 nucleotide to 5 nucleotide ' dangle.The most typically, this strand 3 ' dangle is 1,2,3 or 4 nucleotide.

This siRNA comprises an A sense strand or an A antisense strand, and wherein the length of A chain is that about 15 nucleotide are to about 50 nucleotide; More typically, the length of A chain is that about 18 nucleotide are to about 40 nucleotide; More typically, the length of A chain is that about 20 nucleotide are to about 32 nucleotide; The A chain length is 21,22,23,24,25,26,27,28,29,30 or 31 nucleotide the most typically.

SiRNA of the present invention additionally comprises two or many B chains, here be called, for example B1 and B2, wherein every B chain is complementary to the non-overlapped district of homology A chain, and wherein a B chain (B1) can be separated by otch or one or more nucleotide breach and the 2nd B chain (B2).Depend on that homology A chain is sense strand or antisense strand, every B chain is respectively antisense strand or sense strand.Every B chain described here (B1, B2, etc.) length independently of one another is that about 1 nucleotide is to about 25 nucleotide; Length is that about 4 nucleotide are to about 20 nucleotide more typically; Length is that about 5 nucleotide are to about 16 nucleotide more typically; Length is 6,7,8,9,10,11,12,13,14 or 15 nucleotide the most typically.

In case synthetic A, B1 and B2 chain just are annealed, to form the siRNA molecule.Annealing reaction is determined by complementarity.The one B chain (B1) can be separated by otch or breach and the 2nd B chain (B2).Therefore, remain not pairing and in the siRNA molecule, breach occurs at the nucleotide that (does not therefore have same exogenous nucleotide on the B chain) on the A chain.The size of breach depends between B1 and B2 chain, the quantity of unpaired nucleotide in the A chain.If A chain base and B1 chain and the pairing of B2 chain, otch is formed, and keeps not pairing like this in the zone that does not have nucleotide on the A chain between B1 chain and B2 chain.

In those embodiments that wherein B1 and B2 are separated by breach, described breach is that about 1 nucleotide is to about 25 nucleotide typically; Described more typically breach is that about 1 nucleotide is to about 15 nucleotide; Described more typically breach is that about 1 nucleotide is to about 10 nucleotide; The most described breach is 1,2,3,4,5,6,7,8 or 9 nucleotide.

Each B chain can be independently of one another with 5 ' hydroxyl (that is, 5 '-OH) stop or available 5 ' phosphoric acid (that is, 5 '-PO ₄) stop.Typically, synthetic RNA molecule comprises terminal 3 ' and 5 ' hydroxyl.3 of a B chain therefore, '-OH can be juxtaposed in 5 of next-door neighbour's the 2nd B chain '-OH.In some embodiment, it is comparatively ideal using the 2nd B chain, wherein 5 ' end comprise one 5 '-PO ₄, 3 of such B chain '-OH be juxtaposed in next-door neighbour the 2nd B chain 5 ' PO ₄In this example, rely on PO ₄Group adds to strand (ss) RNA molecule, utilizes the kinase whose catalytic activity of RNA, with the method for knowing in this area and being easy to obtain, realize phosphoric acid is added into 5 of B chain ' end.After kinase reaction was finished, A, B1 and B2 chain were annealed each other, and the B chain is directed to 5 ' B1 pB2 3 ' like this.

Following siRNA molecule has been represented band breach concrete, the indefiniteness example embodiment or otch siRNA molecule, and they are fit to be bonded to one or more as above-mentioned polypeptide here by yoke.

I. gapped duplex siRNA molecule

Certain example gapped duplex siRNA molecule described here be based on a 25-mer have adopted B chain-ordering 5 '-GGAUCUUAUUUCUUCGGAGACAAUG-3 ' (SEQ ID NO:75) and a 27-mer antisense A chain-ordering

3′-TTCCUAGAAUAAAGAAGCCUCUGUUAC-5′(SEQ ID NO：76)。Perhaps exemplary gapped duplex siRNA molecule be based on a 25-mer antisense B chain-ordering 5 '-GGAUCUUAUUUCUUCGGAGACAAUG-3 ' (SEQ ID NO:75) and a 27-mer have adopted A chain-ordering

3′-TTCCUAGAAUAAAGAAGCCUCUGUUAC-5′(SEQ ID NO：76)。

This exemplary siRNA molecule comprises three chain A, B1 and B2 (A:B1B2), wherein B1 and B2 be complementary to A non-overlapped district and also with its formation base pair (bp); Wherein be different from the formed double stranded region of annealing by B2 and A by B1 and the A formed double stranded region of annealing; Wherein the A:B1 duplex is separated by one " breach " in the A chain and A:B2 duplex, described breach is produced by one or more unpaired nucleotide in the A chain, it between A:B1 duplex and A:B2 duplex, and it be different from A, B1 and/or B2 chain wherein one or both 3 ' end on any one or more unpaired nucleotides.

Aspect some of these embodiments, A:B1B2 is by about 14 forming to about 24 base pairs altogether; Wherein A chain (justice is arranged) is that about 19 nucleotide are to about 27 nucleotide; And B1 (antisense) chain and B2 (antisense) chain are that about 1 nucleotide is to about 18 nucleotide independently of one another; Wherein the pattern length of B1+B2 is that about 13 nucleotide are to about 23 nucleotide.

Aspect other of these embodiments, A:B1B2 is by about 16 forming to about 22 base pairs altogether; Wherein A chain (justice is arranged) is that about 19 nucleotide are to about 23 nucleotide; And B1 (antisense) chain and B2 (antisense) chain are that about 1 nucleotide is to about 15 nucleotide independently of one another; Wherein the pattern length of B1+B2 is that about 13 nucleotide are to about 23 nucleotide.

These embodiments further aspect, A:B1B2 is by about 14 forming to about 24 base pairs altogether; Wherein A chain (antisense) is that about 14 nucleotide are to about 27 nucleotide; And B1 (justice is arranged) chain and B2 (justice is arranged) chain are that about 1 nucleotide is to about 18 nucleotide independently of one another; Wherein the pattern length of B1+B2 is that about 18 nucleotide are to about 24 nucleotide.

These embodiments in addition other aspect, A:B1B2 is by about 14 forming to about 22 base pairs altogether; Wherein A chain (antisense) is that about 16 nucleotide are to about 22 nucleotide; And B1 (justice is arranged) chain and B2 (justice is arranged) chain are that about 1 nucleotide is to about 15 nucleotide independently of one another; Wherein the pattern length of B1+B2 is that about 18 nucleotide are to about 22 nucleotide.

Representative siRNA according to these embodiments is shown in the table 2.

Table 2

Representative siRNA

A:B1B2	Sequence identifier	Sequence
			6-nucleoside B1 (justice is arranged) 18-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：77 SEQ ID NO：78 SEQ ID NO：76	5′GGAUCU3′ 5′AUUUCUUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
8-nucleoside B1 (justice is arranged) 16-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：79 SEQ ID NO：80 SEQ ID NO：76	5′GGAUCUUA3′ 5′UUCUUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			10-nucleoside B1 (justice is arranged) 14-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：81 SEQ ID NO：82 SEQ ID NO：76	5′GGAUCUUAUU3′ 5′CUUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
12-nucleoside B1 (justice is arranged) 12-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：83 SEQ ID NO：84 SEQ ID NO：76	5′GGAUCUUAUUUC3′ 5′UCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			14-nucleoside B1 (justice is arranged) 10-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：85 SEQ ID NO：86 SEQ ID NO：76	5′GGAUCUUAUUUCUU3′ 5′GGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
16-nucleoside B1 (justice is arranged) 8-nucleoside B2 (justice is arranged)	SEQ ID NO：87 SEQ ID NO：88	5′GGAUCUUAUUUCUUCG3′ 5′AGACAAUG3′

A:B1B2	Sequence identifier	Sequence
			27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：76	3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
18-nucleoside B1 (justice is arranged) 6-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 1-nucleoside breach	SEQ ID NO：89 SEQ ID NO：90 SEQ ID NO：76	5′GGAUCUUAUUUCUUCGGA3′ 5′ACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			6-nucleoside B1 (justice is arranged) 17-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：91 SEQ ID NO：92 SEQ ID NO：76	5′GGAUCU3′ 5′UUUCUUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
8-nucleoside B1 (justice is arranged) 15-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：93 SEQ ID NO：94 SEQ ID NO：76	5′GGAUCUUA3′ 5′UCUUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			10-nucleoside B1 (justice is arranged) 13-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：95 SEQ ID NO：96 SEQ ID NO：76	5′GGAUCUUAUU3′ 5′UUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
12-nucleoside B1 (justice is arranged) 11-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：97 SEQ ID NO：98 SEQ ID NO：76	5′GGAUCUUAUUUC3′ 5′CGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			14-nucleoside B1 (justice is arranged) 9-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：99 SEQ ID NO：100 SEQ ID NO：76	5′GGAUCUUAUUUCUU3′ 5′GAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
16-nucleoside B1 (justice is arranged) 7-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：101 SEQ ID NO：102 SEQ ID NO：76	5′GGAUCUUAUUUCUUCG3′ 5′GACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			18-nucleoside B1 (justice is arranged)	SEQ ID NO：103	5′GGAUCUUAUUUCUUCGGA3′

A:B1B2	Sequence identifier	Sequence
			5-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 2-nucleoside breach	SEQ ID NO：104 SEQ ID NO：76	5′CAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
6-nucleoside B1 (justice is arranged) 15-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：105 SEQ ID NO：106 SEQ ID NO：76	5′GGAUCU3′ 5′UCUUCGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			8-nucleoside B1 (justice is arranged) 13-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：107 SEQ ID NO：108 SEQ ID NO：76	5′GGAUCUUA3′ 5′UUCGGAGACAAUG 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
10-nucleoside B1 (justice is arranged) 11-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：109 SEQ ID NO：110 SEQ ID NO：76	5′GGAUCUUAUU3′ 5′CGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			12-nucleoside B1 (justice is arranged) 9-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：111 SEQ ID NO：112 SEQ ID NO：76	5′GGAUCUUAUUUC3′ 5′GAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
14-nucleoside B1 (justice is arranged) 7-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：113 SEQ ID NO：114 SEQ ID NO：76	5′GGAUCUUAUUUCUU3′ 5′GACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
			16-nucleoside B1 (justice is arranged) 5-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：115 SEQ ID NO：116 SEQ ID NO：76	5′GGAUCUUAUUUCUUCG3′ 5′CAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′
18-nucleoside B1 (justice is arranged) 3-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 4-nucleoside breach	SEQ ID NO：117 SEQ ID NO：118 SEQ ID NO：76	5′GGAUCUUAUUUCUUCGGA3′ 5′AUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC5′

A:B1B2	Sequence identifier	Sequence
			6-nucleoside B1 (justice is arranged) 13-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：119 SEQ ID NO：120 SEQ ID NO：76	5′GGAUCU3′ 5′UUCGGAGACAAUG 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′
8-nucleoside B1 (justice is arranged) 11-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：121 SEQ ID NO：122 SEQ ID NO：76	5′GGAUCUUA3′ 5′CGGAGACAAUG3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′
			10-nucleoside B1 (justice is arranged) 9-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：123 SEQ ID NO：124 SEQ ID NO：76	5′GGAUCUUAUU 3′ 5′CGGAGACAAUG 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′
12-nucleoside B1 (justice is arranged) 7-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：125 SEQ ID NO：126 SEQ ID NO：76	5′GGAUCUUAUUUC 3′ 5′GACAAUG 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′
			14-nucleoside B1 (justice is arranged) 5-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：127 SEQ ID NO：128 SEQ ID NO：76	5′GGAUCUUAUUUCUU 3′ 5′CAAUG 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′
16-nucleoside B1 (justice is arranged) 3-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：129 SEQ ID NO：130 SEQ ID NO：76	5′GGAUCUUAUUUCUUCG 3′ 5′AUG 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′
			18-nucleoside B1 (justice is arranged) 1-nucleoside B2 (justice is arranged) 27-nucleoside A (antisense) 6-nucleoside breach	SEQ ID NO：131 SEQ ID NO：132 SEQ ID NO：76	5′GGAUCUUAUUUCUUCGGA 3′ 5′G 3′ 3′TTCCUAGAAUAAAGAAGCCUCUGUUAC 5′

What will be appreciated that is, the accurate nucleotide sequence of A, B1 and B2 chain can change, condition is that the siRNA molecule comprises two sections successive base pairs, every section successive base pair is that the breach with at least one nucleotide separates, and this breach is corresponding in the single antisense A chain or single one or more bases of not matching in the adopted A chain are arranged.

Ii. otch duplex siRNA molecule

" p " is illustrated in the otch that has in the RNA chain that 5 ' phosphoric acid is connected to the B2 chain in the sequence of listing below." * " is illustrated in the otch in the RNA chain that does not have 5 ' phosphoric acid to be connected to the B2 chain in the sequence of listing below." p " and " * " goes back the position of indication notch.In all cases, the A chain breach that is kept perfectly (non-incision)～occurs in the A chain will keep the correct series arrangement (because sequence homology) of siRNA molecule.In the sequence of listing below

(black italic) is illustrated in and has ribothymidine (rT) molecule on that position.In the sequence of listing below

(black matrix) is illustrated in and has deoxyribose thymidine (dT) molecule on that position.Successive chain does not promptly have those of breach or otch, is rule below.

1. Dui Zhao dsRNA

Justice is arranged GGAUCUUAUUUCUUCGGAG

-3 ' (SEQ ID NO:133)

Antisense CUCCGAAGAAAUAAGAUCC -3 ' (SEQ ID NO:134)

2. the otch sense strand that does not have 5 ' phosphoric acid

B1 B2

Justice is arranged GGAUCUUAUUU* CUUCGGAG

-3 ' (SEQ ID NO:135 and 136)

Antisense CUCCGAAGAAAUAAGAUCC -3 ' (SEQ ID NO:137)

A

3. the otch sense strand that has 5 ' phosphoric acid

B1 B2

Justice is arranged GGAUCUUAUUUp CUUCGGAG

-3 ' (SEQ ID NO:138 and 139)

Antisense CUCCGAAGAAAUAAGAUCC

-3 ' (SEQ ID NO:140)

A

4. the otch sense strand that has 5 ' phosphoric acid, two chains are all used the rT molecular modification

B1 B2

Justice is arranged GGA

C

A

p C

CGGAG

-3 ' (SEQ ID NO:141 and 142)

Antisense C

CCGAAGAAA

AAGA

CC

-3 ' (SEQ ID NO:143)

A

5. the otch sense strand that does not have 5 ' phosphoric acid, two chains are all used the rT molecular modification

B1 B2

Justice is arranged GGA

A

* C

CGGAG

-3 ' (SEQ ID NO:144 and 145)

Antisense C

CCGAAGAAA

AAGA CC -3 ' (SEQ ID NO:146)

A

6. do not have the otch antisense strand of 5 ' phosphoric acid, antisense strand is used the rT molecular modification fully, and sense strand is not modified (contrast)

A

Justice is arranged GGA

C

A

C CGGAG

-3 ' (SEQ ID NO:147)

Antisense C

CCGAAGAA* A AAGA

CC

-3 ' (SEQ ID NO:148 and 149)

B1 B2

7. have the otch antisense strand of 5 ' phosphoric acid, antisense strand is used the rT molecular modification fully, and sense strand is not modified (contrast)

A

Justice is arranged GGA

C

A

C

CGGAG

-3 ' (SEQ ID NO:150)

Antisense C

CCGAAGAAp A

AAGA

CC

-3 ' (SEQ ID NO:151 and 152)

B1 B2

8.ID: otch S-WT: the duplex that has the otch sense strand

B1 B2

Justice is arranged GGAUCUUAUUU* CUUCGGAG

-3 ' (SEQ ID NO:153 and 154)

Antisense CUCCGAAGAAA UAAGAUCC

-3 ' (SEQ ID NO:155)

A

9.ID: otch S-WT: the duplex that has the otch sense strand; B2 is by 5 ' phosphorylation

B1 B2

Justice is arranged GGAUCUUAUUUp CUUCGGAG

-3 ' (SEQ ID NO:156 and 157)

Antisense CUCCGAAGAAA UAAGAUCC

-3 ' (SEQ ID NO:158)

A

10.ID: otch S-RT: the duplex that has the otch sense strand; Modified by rT fully

B1 B2

Justice is arranged GGA

C

A

* C

CGGAG

-3 ' (SEQ ID NO:159 and 160)

Antisense C CCGAAGAAA AAGA CC

-3 ' (SEQ ID NO:161)

A

11.ID: otch S-RT: the duplex that has the otch sense strand; B2 is by 5 ' phosphorylation; Modified by rT fully

B1 B2

Justice is arranged GGA C

A

p C

CGGAG

-3 ' (SEQ ID NO:162 and 163)

Antisense C

CCGAAGAAA

AAGA

CC

-3 ' (SEQ ID NO:164)

A

12.ID: otch A-RT: the duplex that has the otch antisense strand; Modified by rT fully

A

Justice is arranged GGA C

A

CGGAG -3 ' (SEQ ID NO:165)

Antisense C

CCGAAGAA* A AAGA CC

-3 ' (SEQ ID NO:166 and 34)

B1 B2

13.ID: otch A-RT: the duplex that has the otch antisense strand; B2 is by 5 ' phosphorylation; Modified by rT fully

A

Justice is arranged GGA

C

A

C

CGGAG

-3 ' (SEQ ID NO:38)

Antisense C

CCGAAGAAp A

AAGA

CC -3 ' (SEQ ID NO:39 and 40)

B1 B2

Iii. with the breach or the otch duplex siRNA molecule of connexon closure

In another embodiment, can be (closed) according to breach of the present invention or otch duplex siRNA molecule with connexon molecule " closure ".Resulting molecule should be more stable, promptly has higher T _m, and the degraded in the host cell had more resistance.

According to the present invention, the example of this connexon includes, but not limited to not have the connexon of base, does not for example have the nucleotide of base, the propylene glycol of no base and many little C6-type connexons.

The non-nucleotide connexon can be by the nucleotide of no base, polyethers, polyamine, polyamide, peptide, carbohydrate, lipid, poly-hydrocarbon or other polymerizable compound (for example, Polyethylene Glycol such as have 2 to 100 ethylene glycol unit those) form.Concrete example comprise following described those, Seela and Kaiser etc., Nucleic Acids Res.18:6353,1990 and Nucleic Acids Res.15:3113,1987; Cload and Schepartz, J.Am.Chem.Soc.113:6324,1991; Richardson and Schepartz, J Am.Chem.Soc.113:5109,1991; Ma etc., NucleicAcids Res.21:2585,1993 and Biochemistry 32:1751,1993; Durand etc., NucleicAcids Res.18:6353,1990; McCurdy etc., Nucleosides ﹠amp; Nucleotides 10:287,1991; Jschke etc., Tetrahedron Lett.34:301,1993; Ono etc., Biochemistry 30:9914,1991; Arnold etc., international publication number WO 89/02439; Usman etc., international publication number WO95/06731; Dudycz etc., international publication number WO 95/11910 and Ferentz and Verdine, J.Am.Chem.Soc.113:4000,1991." non-nucleoside acid " means that further can be impregnated in nucleic acid chains replaces one or more nucleotide units, comprises that sugar and/or phosphoric acid replace, and makes any group or the chemical compound of the base reservation that shows their enzymatic activitys.Described group or chemical compound can be no bases, and promptly it does not comprise the nucleotide base that is identified usually, such as adenosine, and guanine, cytosine, uracil or thymus pyrimidine are for example on the C1 position of sugar.

The existence of described connexon can not disturbed the activity of RISC significantly.Preferably, use the RNA that the breach of the present invention that comprises connexon or otch duplex siRNA molecule can cause substantially the same level and disturb, just as when utilize corresponding do not have (non-linked) breach of connection or otch duplex siRNA molecule seen.Most preferably, use the breach of the present invention or the otch duplex siRNA molecule that comprise connexon and can not cause that the corresponding nothing of utilization connects (non-linked) breach or otch duplex siRNA divides the period of the day from 11 p.m. to 1 a.m RNA raising of interference level than working as.

The chemical modification of little inhibition nucleic acid (siNA)

In non-limiting example, chemical modification nucleotide is introduced in the nucleic acid molecules overcoming aspect the potential restriction of exogenous inherent body internal stability of sending of natural RNA molecule and bioavailability a kind of powerful instrument is provided.For example the application of chemical modification nucleic acid molecules can make the therapeutic effect that obtains regulation than the special nucleus acid molecule of low dosage, because the chemical modification nucleic acid molecules trends towards having the longer half-life in serum.In addition, some chemical modification utilizes targeting in specific cells or tissue and/or improve the cellular uptake of nucleic acid molecules and can improve the bioavailability of nucleic acid molecules.Therefore, even the activity of chemical modification nucleic acid molecules is reduced than the natural acid molecule, for example, when than whole RNA nucleic acid molecules, but the gross activity of modified nucleic acid molecule can be greater than this natural molecule because of improving sending of stability and/or molecule.Different with natural unmodified siNA, chemical modification siNA also can make the probability minimum that activates interferon activity in the people.

SiNA molecule described here, the antisense district of siNA molecule of the present invention can comprise one in described antisense district 3 '-end the D2EHDTPA internucleotide linkage.In arbitrary embodiment of described here siNA molecule, the antisense district can comprise about one to about five in described antisense district 5 '-end D2EHDTPA nucleotide bonding.In arbitrary embodiment of described here siNA molecule, 3 of siNA molecule of the present invention '-terminal nucleotide dangles and can be included on ribose, base or the skeleton by the ribonucleotide of chemical modification or deoxyribonucleotide.In arbitrary embodiment of described here siNA molecule, 3 '-terminal nucleotide dangles and can comprise one or more universal base ribonucleotides.In arbitrary embodiment of described here siNA molecule, 3 '-terminal nucleotide dangles and can comprise one or more acyclic nucleoside acid.

For example, in non-limiting example, feature of the present invention is a kind of have in a siNA chain about 1,2,3,4,5,6,7,8 or chemical modification short interfering nucleic acid (siNA) of bonding between the acid of polythio phosphoric acid nucleoside more.In another embodiment, feature of the present invention is a kind of all have separately in two siNA chains about 1,2,3,4,5,6,7,8 or chemical modification short interfering nucleic acid (siNA) of bonding between the acid of polythio phosphoric acid nucleoside more.The D2EHDTPA internucleotide linkage can be present in one of the siNA duplex or two oligonucleotide chains, for example in antisense strand, sense strand or two chains.SiNA molecule of the present invention comprise one or more 3 of sense strand, antisense strand or two chains '-end, 5 '-end or D2EHDTPA internucleotide linkage on both.For example, a kind of exemplary siNA molecule of the present invention comprise about 1 to about 5 or more (for example about 1,2,3,4,5 or more a plurality of) 5 of sense strand, antisense strand or two chains '-continuous D2EHDTPA internucleotide linkage on the end.In another non-limiting example, bonding between the pyrimidine phosphorothioate phosphoric acid nucleoside acid that a kind of exemplary siNA molecule of the present invention comprises is one or more (for example 1,2,3,4,5,6,7,8,9,10 or more a plurality of) in sense strand, antisense strand or two chains.In the another one non-limiting example, the purine D2EHDTPA internucleotide linkage that a kind of exemplary siNA molecule of the present invention comprises is one or more (for example 1,2,3,4,5,6,7,8,9,10 or more a plurality of) in sense strand, antisense strand or two chains.

The siNA molecule can comprise a ringed nucleus acid molecule, wherein the length of this siNA is for (for example having about 18 to about 23, about 18,19,20,21,22 or 23) about 38 of individual base pair to about 70 (for example, about 38,40,45,50,55,60,65 or 70) individual nucleotide wherein should form the dumbbell-shaped structure with about 19 base pairs and 2 rings by the annular oligonucleotide.

Annular siNA molecule contains two ring-type motifs, and wherein one or two annulus of this siNA molecule is biodegradable.For example, annular siNA molecule of the present invention is designed to like this, the vivo degradation of siNA molecule annulus can produce have 3 '-double-stranded siNA molecule that end dangles (for example comprise 3 of about 2 nucleotide '-terminal nucleotide dangles).

Be present in the siNA molecule, preferably in siNA molecule antisense strand and justice and/or antisense are randomly being arranged and modified nucleotide in two chains of justice is arranged, comprise having the modified nucleotide that is similar to natural nucleus ribotide character or characteristic.For example, feature of the present invention is that the siNA molecule comprises (for example having promise gloomy (Northern) conformation, the false rotating ring of promise gloomy (Northern), referring to for example, Saenger, " ultimate principle of nucleic acid structure " (Principles of Nucleic Acid Structure), Springer-Verlag chief editor, 1984) modified nucleotide.Therefore, being present in the siNA molecule of the present invention, preferably in siNA molecule antisense strand of the present invention and justice and/or antisense are randomly being arranged and chemical modification nucleotide in two chains of justice is arranged, is the nuclease-resistant degraded, keeps the ability of mediate rna i simultaneously.Indefiniteness nucleotide example with the gloomy conformation of promise (for example comprises lock nucleic acid (LNA) nucleotide, 2 '-O, 4 '-C-methylene-(D-ribose furyl glycosyl) nucleotide), 2 '-methoxyethoxy (MOE) nucleotide, 2 '-methyl-sulfenyl-ethyl, 2 '-deoxidation-2 '-fluoro nucleotide, 2 '-deoxidation-2 '-chloro nucleotide, 2 '-azido nucleotide and 2 '-the O-methyl nucleotide.

The sense strand of double-stranded siNA molecule can have a distal end cap part for example antisense strand 3 '-end, 5 '-end or 3 ' with 5 '-two ends on inversion deoxidation base portion.

The example of indefiniteness conjugates is included in the U.S. Patent Application Serial Number 10/427,160 (adding here as a reference with its full content) of submissions on April 30th, 2003 such as Vargeese, comprises in its accompanying drawing described conjugates and part.In another embodiment, conjugates covalently is connected to the siNA molecule of chemical modification by biodegradable connexon.In one embodiment, the conjugates molecule be at chemical modification siNA molecule sense strand, antisense strand or two chains 3 '-the end place is connected.In another embodiment, the conjugates molecule be at chemical modification siNA molecule sense strand, antisense strand or two chains 5 '-the end place is connected.In another embodiment, the conjugates molecule be at chemical modification siNA molecule sense strand, antisense strand or two chains 3 '-end with 5 '-two ends of end or their combination in any place are connected.In one embodiment, conjugates molecule of the present invention comprises a kind of like this molecule, and it helps chemical modification siNA molecule to be delivered to living things system for example in the cell.In another embodiment, the conjugates molecule that is connected to chemical modification siNA molecule is the part of Polyethylene Glycol, human serum albumin or a kind of cell receptor that can the mediated cell picked-up.Vargeese etc. have described the conjugated specific adduct molecule example that the present invention anticipated, can be connected to chemical modification siNA molecule on July 10th, 2003 laid-open U.S. Patents application publication number 20030130186 and on June 10th, 2004 laid-open U.S. Patents application publication number 20040110296.The used conjugates type and the conjugates degree of siNA molecule of the present invention can be evaluated, about improving pharmacokinetics situation, bioavailability and/or the stability of siNA construct, keep the active ability of siNA mediate rna i simultaneously.Therefore, those skilled in the art can screen the siNA construct of modifying with various constructs, thus the ability of mediation RNAi in animal model that determines whether that this siNA yoke closes that complex has that the character of having improved keeps simultaneously that this area for example generally knows.

SiNA can be made of nucleotide, non-nucleotide or the mixed nucleotides/non-nucleotide connexon that siNA is had the justice district be connected to siNA antisense district further.In one embodiment, nucleotide linker can be length＞2 nucleotide for example length be the connexon of about 3,4,5,6,7,8,9 or 10 nucleotide.In another embodiment, nucleotide linker can be a kind of aptamer." fit " as used herein or " aptamer " are meant a kind of nucleic acid molecules that is bonded to target molecule specifically, and wherein this nucleic acid molecules has sequence, and this sequence comprises the sequence that a natural setting with it, target molecule are discerned.Perhaps, fit can be a kind of nucleic acid molecules that is attached to target molecule, and this target molecule is not to be bonded to nucleic acid natively.Target molecule can be interested any molecule.For example, fitly be used to be attached to proteic ligand binding domain, prevent native ligand and this protein-interacting by this.This is the example of an indefiniteness, and those skilled in the art will appreciate that use technology generally known in the art can produce at an easy rate other embodiments (referring to, for example, Gold etc., Annu.Rev.Biochem.64:763,1995; Brody and Gold, J.Biotechnol.74:5,2000; Sun, Curr.Opin.Mol.Ther.2:100,2000; Kusser, J.Biotechnol.74:27,2000; Hermann and Patel, Science 287:820,2000; And Jayasena, ClinicalChemistry 45:1628,1999).

The non-nucleotide connexon can be made of (for example, Polyethylene Glycol is such as having those of 2 to 100 ethylene glycol unit) the acid of alkali-free yl nucleosides, polyethers, polyamine, polyamide, peptide, carbohydrate, lipid, poly-hydrocarbon or other polymeric compounds.Concrete example comprise following described those: Seela and Kaiser, Nucleic Acids Res.18:6353,1990 and Nucleic Acids Res.15:3113,1987; Cload and Schepartz, J.Am.Chem.Soc.113:6324,1991; Richardson and Schepartz, J.Am.Chem.Soc.113:5109,1991; Ma etc., NucleicAcids Res.21:2585,1993 and Biochemistry 32:1751,1993; Durand etc., NucleicAcids Res.18:6353,1990; McCurdy etc., Nucleosides ﹠amp; Nucleotides 10:287,1991; Jschke etc., Tetrahedron Lett.34:301,1993; Ono etc., Biochemistry 30:9914,1991; Arnold etc., international publication number WO 89/02439; Usman etc., international publication number WO95/06731; Dudycz etc., international publication number WO 95/11910 and Ferentz and Verdine, J.Am.Chem.Soc.113:4000,1991." non-nucleoside acid " is meant any group or chemical compound further, can be incorporated into to replace one or more nucleotide units in the nucleic acid chains, comprises that sugar and/or phosphoric acid replace, and makes the base that keeps show their enzymatic activity.Described group or chemical compound can be no bases, and promptly it does not contain common nucleotide base, for example on the C1 position of sugar such as adenosine, guanine, cytosine, uracil or thymus pyrimidine.

Use the compositions of the polypeptide/siNA conjugates that comprises that the raising polynucleotide are sent

Polypeptide/siNA conjugates that the present invention sends raising can be used to treat as discussed herein or other disease known in the art or diseases.In order to treat special disease or disease, polypeptide/siNA conjugates that the present invention sends raising can be applied to the patient or can be applied to is other obvious suitable cells to those skilled in the art, under the condition that is suitable for treating respectively or with the combined ground of more than one chemical compounds.

For example, polypeptide/siNA conjugates of sending raising described here can be used to and other known treatments and/or therapeutic agent associating, so that treat various diseases, particularly viral infection.Polypeptide/siNA conjugates non-limiting example linked together, the other treatment agent that can be easy to send with the present invention raising comprises, for example, and the nucleic acid molecules of enzyme; Other structure nucleic acid molecules; Antisense, trapping thing or fit nucleic acid molecules; Antibody is such as monoclonal antibody; Micromolecule; And other organic and/or inorganic chemical compounds, comprise metal, salt and ion.

Therefore, the invention describes at a kind of acceptable carrier and comprise that one or more send the compositions of the polypeptide of raising/siNA conjugates in such as stabilizing agent, buffer agent etc.Described polypeptide/siNA conjugates of sending raising can with or not with stabilizing agent, buffer agent etc., be applied to the patient in the mode of arbitrary standard, so that form a kind of compositions that is suitable for treating.When liposome delivery mechanism is used in expectation, can follow the standard scheme that forms liposome.Compositions of the present invention also can be mixed with preparation and as tablet, capsule or the elixir of oral administration, the suppository of rectally, the sterile solution of injectable administration and suspension, arbitraryly with or not and other chemical compounds known in the art.Polypeptide/siNA conjugates preparation of sending raising comprises the salt of above-claimed cpd, for example the salt of all example hydrochloric acids of acid-addition salts, hydrobromic acid, acetic acid and benzenesulfonic acid.

Pharmaceutical composition or preparation are meant for example to be fit to use systemic administration to the compositions or the preparation of the form of cell or patient such as philtrum.The form that is fit to partly, depends on the approach of using or entering, and is for example oral, transdermal or by injection.These forms should not stop described compositions or preparation to reach target cell (that is, the expectation of electronegative nucleic acid send cell).For example, the pharmaceutical composition that is injected in the blood flow should be soluble.Other factors are known in the art, and comprise and prevent that compositions or preparation from bringing into play considering such as toxicity and form of its effect.

Independent reporter gene expression construct can be sent the polypeptide/siNA conjugates cotransfection of raising with one or more.Polypeptide/siNA conjugates that particular delivery improves reduces each and expects the ability of genetic mutation expression, can determine by the reporter gene activity of detection that contrasts the siNA of modification transfectional cell personal or of no use.

The method that nucleic acid molecules is sent is described in Akhtar etc., Trends Cell Bio.2:139,1992; " delivery strategies of antisense strategy agent (Delivery Strategies for AntisenseOligonucleotide Therapeutics) " (chief editor: Akhtar, 1995); Maurer etc., Mol.Membr.Biol 16:129-140,1999; Hofland and Huang, Handbook Exp.Pharmacol.137:165-192,1999; And Lee etc., ACS Symp.Ser.752:184-192,2000.Sullivan etc., PCT WO 94/02595 has further described the universal method of sending the enzymatic nucleic acid molecule.These schemes all can be used for sending of in fact arbitrary nucleic acid molecules.

As used herein, term " systemic administration " is meant the interior systematicness absorption of the body that comprises the blood flow Chinese medicine or gathers the whole whole body that distributes subsequently.Cause that the route of administration that systematicness absorbs comprises, is not limited to: in venous, subcutaneous, endoperitoneal, suction, oral, the lung with intramuscular.In these route of administration every kind all with desired electronegative polymer, for example nucleic acid is exposed to enterable illing tissue.Medicine enters the function that circulation speed has been proved to be molecular weight or size.

Nucleic acid molecules can be applied to cell, with known variety of processes to those skilled in the art, but is not limited to, and is encapsulated in the liposome; With iontherapy; Or by mixing other media body such as hydrogel, cyclodextrin, biodegradable Nano capsule and bioadhesive microsphere; Or through protein carrier (O ' Hare and Normand, International PCT publication No. WO 00/53722).Perhaps, nucleic acid/vehicle combination can be sent partly through direct injection or through using infusion pump.The direct injection of nucleic acid molecules of the present invention can carry out, no matter be subcutaneous, intramuscular or Intradermal, by application standard syringe needle and syringe method, or with the needleless technology such as at Conry etc., Clin.CancerRes.5:2330-2337,1999 and Barry etc., those described in the International PCT publication No. WO 99/31262.

Here disclosed polypeptide/siNA conjugates of sending raising can be used as medicament.Medicament prevention, the appearance of regulating morbid state among the patient or the morbid state among the treatment patient (with mitigation symptoms to a certain degree, preferably whole symptoms).

As used herein, phrase " pharmaceutically acceptable preparation " is meant and comprises that permission nucleic acid molecules of the present invention is distributed in compositions or the preparation that is suitable for most in their desired active physical locations effectively.The example that is suitable for being mixed with nucleic acid molecules of the present invention the indefiniteness reagent of preparation comprises: P-glycoprotein inhibitors (such as Pluronic P85), it can improve medicine and enter CNS (Jolliet-Riant and Tillement, Fundam.Clin.Pharmacol.13:16-26,1999); Biodegradable polymer, such as be implanted at brain poly-(DL-lactide-Acetic acid, hydroxy-, bimol. cyclic ester) altogether microsphere (Emerich etc., Cell Transplant 8:47-58,1999) that the back continues release delivery (Alkermes Inc., Cambridge, Mass); And the nanoparticle of medicine carrying, such as by those of paracyanogen base butyl acrylate manufacturing, but their delivering drugs are by blood brain barrier and can change neuronal uptake mechanism (ProgNeuropsychopharmacol Biol Psychiatry 23:941-949,1999).

Application comprises that the liposome of The compounds of this invention or other drug carrier can make the medicine localization potentially, for example, and in some types of organization, in the tissue such as reticuloendothelial system (RES).Similarly, can promote that medicine is useful with the mutually associating Liposomal formulation in the surface of cell such as lymphocyte and macrophage.By the specificity and the lymphocyte immunity identification abnormal cell that utilize macrophage, this method can provide the send raising of medicine to target cell.

Other non-limiting examples of nucleic acid molecules delivery strategies of the present invention are included in Boado etc., J.Pharm.Sci 87:1308-1315,1998; Tyler etc., FEBS Lett.421:280-284,1999; Pardridge etc., PNAS USA 92:5592-5596,1995; Boado, Adv.Drug Delivery Rev.15:73-107,1995; Aldrian-Herrada etc., Nucleic Acids Res.26:4910-4916,1998; And Tyler etc., PNAS USA.96:7053-7058, the material described in 1999.

Additional features of the present invention is the application that comprises the finishing liposome composition, and described liposome contains poly-(ethylene glycol) lipid (PEG-modifies, or long liposome or the hidden liposome of circulation time).These preparations provide a kind of method that medicine gathers that increases in target tissue.This class medicine carrier is by monokaryon phagocyte system (MPS or RES) and anti-opsonic action and elimination, and the tissue that makes by this that blood circulation time is longer and improve entrapped drug exposes.Lasic etc., Chem.Rev.95:2601-2627,1995; Ishiwata etc., Chem.Pharm.Bull.43:1005-1011,1995.This liposome has been proved to be to blend at home and abroad by the target tissue at revascularization and has caught and optionally gather in tumor.Lasic etc., Science 267:1275-1276,1995; Oku etc., Biochim.Biophys.Acta 1238:86-90,1995.The long liposome of circulation time has improved pharmacokinetics and the pharmacodynamics of DNA and RNA, particularly than the known conventional cationic-liposome that gathers in the MPS tissue.Liu etc., J.Biol.Chem.42:24864-24870,1995; Choi etc., International PCT publication No. WO 96/10391; Ansell etc., International PCT publication No. WO 96/10390; With Holland etc., International PCT publication No. WO 96/10392.The long liposome of circulation time also may protect medicine not to be subjected to nuclease degradation to a greater degree, than cationic-liposome, because they are avoided at the aggressive MPS tissue of metabolism as liver and the poly-ability of splenic dyspepsia.

The present invention also is included as the compositions of preserving or using and prepare, and they are included in the desired chemical compound of the pharmaceutically effective dose in pharmaceutically acceptable carrier or the diluent.Carrier accepted or diluent that treatment is used are to know in the pharmaceutical field, and been described in, for example, " Remington ' sPharmaceutical Sciences ", Mack publishing company, A.R.Gennaro chief editor, 1985 years.For example, the present invention can provide antiseptic, stabilizing agent, stain and flavoring agent.These comprise sodium benzoate, sorbic acid and p-Hydroxybenzoate.In addition, the present invention also can use antioxidant and suspending agent.

Pharmaceutically effectively dosage is the required dosage of prevention, the generation that suppresses morbid state or treatment morbid state (with relief of symptoms to a certain degree, preferably all symptoms).Pharmaceutically effective dose depend on disease type, compositions for use, the approach of administration, the mammiferous type for the treatment of, consider specific mammiferous physical trait, the common medicine that exists and other factors that the technical staff can recognize in medical domain.Usually, quantity is that the active component of 0.1mg/kg to 100mg/kg body weight/day is applied, and depends on the effectiveness of electronegative polymer.

The present invention also is included as the compositions of preserving or using and prepare, and they are included in the desired chemical compound of the pharmaceutically effective dose in pharmaceutically acceptable carrier or the diluent.Additional or the complementarity method of nucleic acid delivery molecule is described in, for example, and Akhtar etc., Trends Cell Bio.2:139,1992; " delivery strategies of antisense strategy agent ", Akhtar chief editor, 1995; Maurer etc., Mol Membr.Biol.16:129-140,1999; Hofland and Huang, Handb.Exp.Pharmacol.137:165-192,1999; And Lee etc., ACS Symp.Ser.752:184-192,2000.Sullivan etc., International PCT publication No. WO 94/02595 has further described the conventional method of sending the enzymatic nucleic acid molecule.These schemes can be utilized so that the sending of being considered among additional or complementary the present invention of nucleic acid molecules in fact arbitrarily.

The polypeptide that nucleic acid molecules and raising polynucleotide are sent can be applied to cell with the whole bag of tricks well known by persons skilled in the art, include but not limited to, using in preparation, said preparation only comprises siNA and improves the polypeptide that polynucleotide are sent, perhaps it comprises the component that one or more are extra further, such as pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifying agent, buffer agent, stabilizing agent, antiseptic etc.In certain embodiments, the polypeptide that siNA and/or raising polynucleotide are sent can be encapsulated in the liposome, uses with iontherapy; Or mix other media thing such as hydrogel, cyclodextrin, biodegradable Nano capsule, bioadhesive microsphere or protein carrier (referring to, for example, O ' Hare and Normand, International PCT publication No. WO 00/53722).Perhaps, nucleic acid/peptide/vehicle combination can be sent partly through direct injection or through using infusion pump.The direct injection of nucleic acid molecules of the present invention can carry out, no matter be subcutaneous, intramuscular or Intradermal, by application standard syringe needle and syringe method, or with the needleless technology such as at Conry etc., Clin.CancerRes.5:2330-2337,1999 and Barry etc., those described in the International PCT publication No. WO 99/31262.

Compositions of the present invention can be used as medicament effectively.Medicament prevention, regulate the generation or the seriousness of morbid state among the patient or other unfavorable situations, perhaps treat among the patient morbid state or other unfavorable situations (can detect or measurable degree is alleviated one or more symptoms).

Therefore, in other embodiments, the invention provides pharmaceutical composition and method, feature is existence or uses one or more Polynucleotides, one or more siNA mainly, with improve polypeptides in combination that polynucleotide send, compound or yoke closes, and randomly is mixed with preparation with pharmaceutically acceptable carrier such as diluent, stabilizing agent, buffer agent etc.

The present invention has satisfied other purpose and advantage by short interfering nucleic acid (siNA) molecule is provided, the relevant gene expression of special disease state or other unfavorable situations among this siNA molecular regulation curee.Typically, the siNA targeting is in a kind of gene to improve the standard and to be expressed, and the level of this raising is as a kind of cause or the contribution factor relevant with curee's morbid state or unfavorable situation.Under this situation, siNA reduces this expression of gene effectively to the level of preventing, alleviate or reduce one or more associated disease symptom seriousness or recurrence.Perhaps, needn't be as the result of disease or unfavorable situation or consequence and the various unique disease model that is enhanced for target gene expression, but the target gene downward modulation causes the therapeutic result by reducing expression of gene (that is, reducing the selected mRNA of target gene and/or the level of protein product).Perhaps, siNA of the present invention can be by targeting, so that reduce an expression of gene, this can cause the rise of " downstream " gene, should " downstream " gene expression be to be regulated by a kind of product or active the bearing of target gene.

In exemplary, the compositions and methods of the invention are useful, regulate tumor necrosis factor-alpha (TNF-α) thereby the symptom of treatment or prevention rheumatoid arthritis (RA) as the therapeutic instrument.Under this situation, the present invention provides further about using the small nucleic acids molecule, disturbing (RNAi) to regulate useful chemical compound, compositions and the method for expression and active aspect of TNF-α with RNA.In more detailed embodiment, the invention provides small nucleic acids molecule such as short interfering nucleic acid (siNA), short interfering rna (siRNA), double-stranded RNA (dsRNA), microRNA (mRNA), and short hairpin RNA (shRNA) molecule, with and related methods, thereby they are effective aspect the RA symptom in regulating TNF-α and/or TNF-α gene expression prevention or releasing mammal curee.In these and associated treatment compositions and method; for example by the resistance of enhancing to the nuclease vivo degradation is provided; and/or by improving the picked-up of cell, the application of chemical modification siNA usually can improve the character of modification siNA, than the character of natural siNA molecule.As being easy to determine that according to disclosed content here the useful siNA with multiple chemical modification can keep their RNAi activity.Therefore, siNA molecule of the present invention provides useful reagent and the method that various therapeutic, diagnostic, target affirmation, genome research, genetic engineering and medicine genome are used.

This siNA of the present invention can pass through any way administration, as with transdermal means or local injection (as at plaque psoriasis site local injection with the treatment psoriasis, or be expelled to the patient's who suffers from psoriatic arthritis or RA intraarticular).In more detailed embodiment, the invention provides preparation and method siNA, the inflammatory states that can reduce TNF-α RNA effectively and therefore reduce or prevent one or more TNF-α to be correlated with in order to the mRNA of the directed anti-TNF-α of administering therapeutic effective dose.Comparable method and composition is provided, the selected relevant gene of morbid state in their targeting one or more different and the animal subject, but comprise many known its expression abnormal ascending, can be used as in many genes of the cause of disease relevant or influence factor any one with selected morbid state.

SiNA/ raising polynucleotide of the present invention are sent mixtures of polypeptides and can be united use with other standard care method that is used for the target morbid state, for example with antagonism inflammatory diseases such as RA or the effective therapeutic agent associating of psoriasis.Useful and example effective agents under this background comprises nonsteroidal antiinflammatory drug (NSAID), methotrexate, gold compound, Beracilline, antimalarial, sulfasalazine, glucocorticoid and other TNF-α neutralization reagent such as infliximab and entracept.

Electronegative polynucleotide of the present invention (as RNA or DNA) can any standard mode to patient's dispenser, can with or do not form pharmaceutical compositions with stabilizing agent, buffer agent etc.When hope utilizes liposome delivery mechanism, can follow the standard test method that forms liposome.Compositions of the present invention is tablet, capsule or the elixir to be used for oral administration also, is used for the suppository of rectally, and the sterile solution of injectable administration, suspension or other are composition forms preparation known in the art and use.

The present invention also comprises the acceptable preparation of the pharmacy of compositions described herein.These preparations comprise the salt of above chemical compound, for example the salt of acid-addition salts example hydrochloric acid, hydrobromic acid, acetic acid and benzenesulfonic acid.

Pharmacology's compositions or preparation refer to that compositions or preparation take to be fit to the form of dispenser, for example comprise for example system's dispenser of people to cell or patient.Suitable form depends in part on occupation mode or route of entry, for example oral cavity, transdermal or by injection.This form should not hinder compositions or preparation arrives target cell (for example expecting the cell of electronegative nucleic acid to its administration).For example, the pharmacology's compositions that is expelled in the blood flow should be soluble.Other factors is well known, comprises and should consider toxicity.

" being administered systemically " means system's absorption or accumulation in the body of medicine in blood flow, distributes to whole body subsequently.Can cause that the route of administration that system absorbs includes but not limited to: in vein, subcutaneous, intraperitoneal, suction, oral cavity, the lung and intramuscular.Every kind in these route of administration all can be exposed to come-at-able illing tissue with the electronegative polymer such as the nucleic acid of expectation.The speed that known drug enters blood circulation is the function of its molecular weight or size.Utilize liposome or other to comprise the pharmaceutical carrier of the The compounds of this invention medicine that can localize potentially, for example in particular tissue type, as reticuloendothelial system (RES).It also is useful can making medicine and the link coupled Liposomal formulation of for example lymph and macrophage surface.The method can strengthen sending to target cell by utilizing macrophage and lymphocytic immunity to discern the specificity advantage of improper cell such as cancer cell.

" the acceptable preparation of pharmacy " means compositions or the preparation that nucleic acid molecules of the present invention can effectively be distributed to the most suitable their required active body area.Be fit to comprise as the non-limitative example of the reagent of preparation jointly: P-glycoprotein inhibitors (as PluronicP85) with nucleic acid molecules of the present invention, it can promote medicine to enter CNS (Jolliet-Riant and Tillement, Fundam.Clin.Pharmacol.13:16-26,1999); Biodegradable polymer, as poly-(DL-lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer microsphere (Emerich, the D.F. etc. of the lasting release delivery after being used for brain and being implanted into, Cell Transplant 8:47-58,1999) (Alkermes Inc., Cambridge, Mass); The nano-particle of medicine carrying, as the granule of being made by PBCA, they can pass the blood brain barrier administration and can change neural mechanism of absorption (Prog.Neuropsychopharmacol Biol.Psychiatry 23:941-949,1999).Other non-limitative example that is used for the administration strategy of nucleic acid molecule of the present invention comprises the interior description of following document: Boado etc., J.Pharm.Sci.87:1308-1315,1998; Tyler etc., FEBSLett.421:280-284,1999; Pardridge etc., PNAS USA.92:5592-5596,1995; Boado, Adv.Drug Delivery Rev.15:73-107,1995; Aldrian-Herrada etc., Nucleic Acids Res.26:4910-4916,1998; With Tyler etc., PNAS USA.96:7053-7058,1999.

The present invention also is included as storage or administration and the compositions for preparing, and it is included in the required compound of the pharmacy effective dose in acceptable carrier of pharmacy or the diluent.The acceptable carrier and the diluent that are used for the treatment of purposes are that the pharmaceutics field is known, for example at Remington ' sPharmaceutical Sciences, Mack publishing company, A.R.Gennaro compiles, description is arranged in 1985, incorporate this paper by reference at this.For example, may provide antiseptic, stabilizing agent, dyestuff and flavour enhancer.These comprise sodium benzoate, sorbic acid and p-Hydroxybenzoate.In addition, also may use antioxidant and suspending agent.

Pharmacy effective dose is meant prevention, suppress the appearance of morbid state or to the needed dosage of its treatment (to a certain degree, preferably is all symptoms with remission).Pharmacy effective dose depends on disease type, composition therefor, route of administration, the mammiferous type for the treatment of, the specific mammiferous physiological feature of being considered, parallel medication and is familiar with the other factors that the people of medical industries knows.According to tiring of electronegative polymer, generally use the amount of 0.1mg/kg to 100mg/kg body weight/day active ingredient.

Raising of the present invention send polypeptide/siNA conjugates and preparation thereof can by suck or spraying through the oral cavity, part, intestinal medicine for external use, or to contain the dosage unit preparations per rectum administration of the traditional acceptable carrier of non-toxicity pharmacy, adjuvant and/or excipient.The term parenteral of Shi Yonging comprises (for example vein), intramuscular or intrathecal injection or infusion or similar techniques in percutaneous, subcutaneous, the blood vessel outward herein.In addition, provide a kind of pharmaceutical preparation, it comprises nucleic acid molecules of the present invention and the acceptable carrier of a kind of pharmacy.One or more nucleic acid molecules of the present invention can with one or more acceptable carriers of non-toxicity pharmacy and/or diluent and/or adjuvant associating, and if needed with other active ingredient associating.The pharmaceutical composition that contains nucleic acid molecules of the present invention can take to be fit to the form of oral cavity medicine, but for example be tablet, buccal tablet, lozenge, water or oil suspension dispersed powders or electuary, emulsion, firmly or soft capsule, syrup or elixir.

The method preparation that the compositions used in the oral cavity can be known according to any industry is done in plan, is used to produce pharmaceutical composition, and this compositions can contain one or more sweeting agents, flavour enhancer, coloring agent or antiseptic, so that top grade and agreeable to the taste medicament prepared product to be provided.Tablet contains the excipient that active ingredient adds the acceptable suitable tablet manufacturing of atoxic pharmacy.These excipient can be for example inert diluent, as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; Granulation agent and disintegrating agent, for example corn starch or alginic acid; Binding agent, for example starch, gelatin or arabic gum; Lubricant, for example magnesium stearate, stearic acid or Pulvis Talci.Tablet is coating or use known technology coatings not.Available in some cases known technology carries out certain coating, postponing disintegrate and the absorption in gastrointestinal tract, thereby provides the continuous action of long term.For example, can utilize time-delay material such as glyceryl monostearate or glycerol distearate.

The preparation that is used for using in the oral cavity also can adopt the hard gelatin capsule form, active ingredient has herein mixed inert diluent, for example calcium carbonate, calcium phosphate or Kaolin, or with the Perle form, active ingredient has herein mixed water or oily medium, for example Oleum Arachidis hypogaeae semen, liquid paraffin or olive oil.

Suspension comprises the suitable excipient that has mixed active ingredient and the production of suitable suspension.This excipient is a suspending agent, for example sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl emthylcellulose, sodium alginate, polyvinylpyrrolidone, tragakanta and arabic gum; Dispersant or wetting agent, can be natural phospholipid, the condensation product of lecithin or epoxyalkane and fatty acid for example, as polyoxyethylene 8 stearate, or the condensation substance of oxirane and long-chain fatty alcohol, as 17 ethylene oxy hexadecanol (heptadecaethyleneoxycetanol), or the condensation substance such as the octadecanoic acid ester of polyethylene glycol of oxirane and fatty acid and the deutero-partial ester of hexitol, or oxirane and from the condensation substance of fatty acid and the deutero-partial ester of hexitan, for example polyethylene Arlacel-80.Suspension also can contain one or more antiseptic, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agent, one or more flavour enhancer, and one or more sweeting agents, for example sucrose or glucide.

Oily suspensions can be prepared by suspension active ingredient in vegetable oil, for example in Oleum Arachidis hypogaeae semen, olive oil, Oleum sesami or Oleum Cocois, or in mineral oil, as liquid paraffin.Oily suspensions can contain thickening agent, for example Cera Flava, hard paraffin or hexadecanol.Can add sweeting agent and flavour enhancer, so that agreeable to the taste oral cavity prepared product to be provided.These compositionss can add antioxidant such as ascorbic acid with anticorrosion.

The dispersible powder and the granule that are suitable for supending are by add entry in active ingredient and dispersant or wetting agent, suspending agent and one or more antiseptic so that they mix.Suitable dispersant or wetting agent or suspending agent are above having example.Also can add other excipient, for example sweeting agent, flavour enhancer and coloring agent.

Pharmaceutical composition of the present invention also can be O/w emulsion.Oil phase can be vegetable oil or mineral oil or their mixture.Suitable emulsion can be natural gum, as arabic gum or tragakanta, natural phospholipid, for example Semen sojae atricolor, lecithin and from fatty acid and deutero-ester of hexitol or partial ester, acid anhydride, Arlacel-80 for example, and the condensation substance of described partial ester and oxirane, for example polyoxyethylene sorbitan monooleate dehydration.Emulsion also can contain sweeting agent and flavour enhancer.

Syrup and elixir can be prepared with sweeting agent, for example glycerol, propylene glycol, sorbitol, glucose or sucrose.This preparation also can contain demulcent, antiseptic, flavour enhancer and coloring agent.Pharmaceutical composition can be the injectable liquids or the oleagenous suspension of sterilization.This suspension can utilize suitable dispersant mentioned above or wetting agent and suspending agent preparation according to the known technology in this field.The injectable prepared product of sterilization also can be the Injectable solution or the suspension of the sterilization in outside atoxic intestinal acceptable diluent or the solvent, for example is 1,3 butylene glycol solution.Acceptable vehicle thing that may use and solvent are water, Ringer ' s solution and isotonic sodium chlorrde solution.In addition, aseptic nonvolatile oil is used as solvent or suspension media traditionally.Any gentle nonvolatile oil all can be used for this purpose, comprises synthetic monoglyceride or diglyceride.In addition, fatty acid such as oleic acid can be used for preparing the injectable thing.

Polypeptide/siNA conjugates is sent in raising of the present disclosure also can suppository form dispenser, for example the rectum dispenser of medicine.This compositions can be by hybrid medicine and suitable non-irritating excipient preparation, and this excipient should be solid-state at normal temperatures, and is liquid under the temperature of rectum, therefore can melt in rectum to discharge medicine.This class material comprises cocoa butter and Polyethylene Glycol.

Polypeptide/siNA conjugates is sent in raising of the present disclosure can dispenser outside the intestinal in sterile media.Can be according to used vehicle and concentration with pharmaceutical suspension or be dissolved in the vehicle.Advantageously, adjuvant such as local anaesthetics, antiseptic and buffer agent can be dissolved in vehicle.

Dosage level from about 0.1mg to the every kg body weight of about 140mg every day is effectively (the about 0.5mg of each patient to about 7g every day) for treatment of diseases mentioned above.Be used for manufacture order dosage form when packing, can change with the amount of the active ingredient of carrier combination of materials ad hoc fashion according to treatment host and dispenser.Dosage unit form generally contains the active ingredient of about 1mg to about 500mg.

The given dose level that becomes known for any particular patient depends on series of factors, comprises the given activity, age, body weight, general health situation, sex, diet, administration time, route of administration, excretion rate of used chemical compound, the seriousness of the medication combined and specified disease for the treatment of.

When being used for inhuman animals administer, also compositions can be added in animal feed or the drinking water.Preparing animal fodder and drinking water composition are more convenient, can make animal take the compositions of therapeutics appropriate amount with its diet.Compositions is made as to be used for the premix material that adds to feedstuff and drinking water also more convenient.

Improve and send polypeptide/siNA conjugates other medicines chemical compound also capable of being combined to patient's dispenser, to improve total therapeutic effect.Use certain disease of multiple compounds for treating can improve beneficial effect and reduce side effect.

In one embodiment, inventive compositions is fit to raising is sent polypeptide/siNA conjugates dispenser in the special cells type, as liver cell.For example, asialoglycoprotein receptor (ASGPr) is that liver cell is exclusive, and it is in conjunction with side chain galactose-terminal glycoprotein, as asialoglycoprotein mucin (ASOR).Can utilize the nuclease-resistant group for example 2 '-amino, 2 '-C-pi-allyl, 2 '-fluorine, 2 '-O-methyl, 2 '-H modifies WuThe siNA and to its meticulous modification, to strengthen its stability.(see summary Usman and Cedergren, TIBS 17:34,1992; Usman etc., Nucleic AcidsSymp.Ser.31:163,1994).The SiNA construct can utilize conventional method to pass through the gel electrophoresis purification or pass through high pressure liquid chromatography purification and resuspended in water.

The nucleic acid molecules with modification (base, sugar and/or phosphate ester) of chemosynthesis can prevent that it from by serum ribonucleic acid enzymatic degradation, improving it and tiring.For example see Eckstein etc., international publication number WO92/07065; Perrault etc., Nature 344:565,1990; Pieken etc., Science253:314,1991; Usman and Cedergren, Trends inBiochem.Sci.17:334,1992; Usman etc., international publication number WO 93/15187; With Rossi etc., international publication number WO 91/03162; Sproat, U.S. Patent number 5,334,711; Gold etc., U.S. Patent number 6,300,074.The various chemical modifications that all above citing documents are described all can be used for base, phosphate ester and/or the sugar moieties of nucleic acid molecule described herein.

This area has several examples to describe sugar, base and phosphate ester modified introduces nucleic acid molecules and can significantly strengthen it to the stability of nuclease with tire.For example,, can strengthen the stability of adorned oligonucleotide and/or strengthen its biologic activity by with nuclease resistance base group modification, for example 2 '-amino, 2 '-C-pi-allyl, 2 '-fluorine, 2 '-O-methyl, 2 '-H, nucleotide base modify.See summary Usman and Cedergren, TIBS 17:34,1992; Usman, etc., Nucleic Acids Symp.Ser.31:163,1994; Burgin etc., Biochemistry 35:14090,1996.The sugar-modified of nucleic acid molecule has a detailed description in this area.See Eckstein etc., international publication PCT WO92/07065; Perrault etc., Nature 344:565-568,1990; Pieken etc., Science253:314-317,1991; Usman and Cedergren, Trends in Biochem.Sci.17:334-339,1992; Usman etc., international publication PCT WO 93/15187; Sproat, U.S. Patent number 5,334,711 and Beigelman etc., J.Biol.Chem.270:25702,1995; Beigelman etc., International PCT publication No. WO97/26270; Beigelman etc., U.S. Patent number 5,716,824; Usman etc., U.S. Patent number 5,627,053; Woolf etc., International PCT publication No. WO 98/13526; Thompson etc., Karpeisky etc., Tetrahedron Lett.39:1131,1998; Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55,1998; Verma and Eckstein, Annu.Rev.Biochem.67:99-134,1998; With Burlina etc., Bioorg.Med.Chem.5:1999-2010,1997.These publications have been described and sugar, base and/or phosphate ester trim or analog be coupled in the nucleic acid molecules and do not changed conventional method and the strategy that the site of catalytic action is determined.For these contents of teaching, also can utilize similar remodeling method described herein to modify siNA nucleic acid molecules of the present invention, as long as siNA promotes the ability of RNAi not to be subjected to obvious inhibition in cell.

Though with thiophosphate, phosphorodithioate and/or 5 '-the methylphosphonic acid key carries out chemical modification to key between oligonucleotide nucleotide and can improve stability, excessive grooming can cause toxicity or active reduction the to a certain degree.Therefore, when designing nucleic acid divides the period of the day from 11 p.m. to 1 a.m, should be with the minimum numberization of key between these nucleotide.The minimizing of these key concentration can reduce toxicity, causes the enhancing of tiring.Wu，J.Biol.Chem.262：4429-4432，1987。This glycoprotein or synthetic glycoconjugates depend on the degree of branching of oligonucleotide chain strongly to the affinity of receptors bind, for example, the binding ratio double antenna (biatennary) of triantennary (triatennary) structure or single antenna (monoatennary) chain have bigger affinity.Baenziger and Fiete, Cell 22:611-620,1980 and Connolly etc., J.Biol.Chem.257:939-945,1982.Lee and Lee have obtained this high specific by utilizing N-acetyl-D-galactosamine as sugar moieties, and itself and galactose ratio have the high-affinity to receptor.Glycoconjugate J.4：317-328，1987。This " constellation effect " once described in the combination of terminal glycoprotein of mannose or glycoconjugates with in absorbing.Ponpipom etc., J.Med.Chem.24:1388-1395,1981.Utilizing galactose and galactosamine nucleotide conjugates that xenobiontics is striden the film transportation can provide a kind of targeting application method to be used for the treatment of hepatopathy, infects or hepatocarcinoma as HBV.The use of bioconjugates also can reduce the dosage of the required medical compounds of treatment.Can be further by utilizing biological nucleic acid conjugates adjustment of treatment bioavailability of the present invention, pharmacodynamics and pharmacokinetic parameter, to obtain the higher specificity of these molecules.

In one embodiment, the present invention is characterized as the siNA molecule of the modification of tool phosphate ester backbone modification, and it comprises one or more thiophosphates, phosphorodithioate, methylphosphonic acid, phosphotriester, morpholine, amide carbamate, carboxymethyl, acetyl amide (acetamidate), polyamide, sulfonate, sulfonamide, sulfamate, modification acetal (formacetal), sulfo-modification acetal (formacetal) and/or alkane silicon substitute.See below to obtain summary: Hunziker and Leuman about the oligonucleotide framework modification, " Nucleic Acid Analogues:Synthesis andProperties, in Modem Synthetic Methods, " VCH, 331-417,1995 and Mesmaeker etc., " Novel Backbone Replacement for Oligonucleotides; inCarbohydrate Modifications in Antisense Research; " ACS, 24-39,1994.

The method of sending nucleic acid molecule has description: Akhtar etc., Trends CellBio.2:139,1992 in following document; Delivery Strategies for Antisense OligonucleotideTherapeutics, Akhtar compiles, and 1995; Maurer, etc., Mol, Membr.Biol.16:129-140,1999; Hofland and Huang, Handb.Exp.Pharmacol.137:165-192,1999; With Lee etc., ACS Symp.Ser.752:184-192,2000.Beigelman etc., U.S. Patent number 6,395,713 and Sullivan etc., PCT WO 94/02595 has further described the conventional method of sending nucleic acid molecule.These experimental programs can be used for sending of in fact any nucleic acid molecules.Can nucleic acid molecules be administered in the cell by the method that many personnel that are familiar with industry technology know, include but not limited to the liposome methods of packing into, pass through iontophoresis, or pass through and other vehicle combination, (example is seen Gonzalez etc. as biodegradable polymer, hydrogel, cyclodextrin, Bioconjugate Chem.10:1068-1074,1999; Wang etc., International PCT publication No. WO 03/47518 and WO03/46185), (example is seen U.S. Patent number 6 for polylactic acid-polyglycolic acid copolymer (PLGA) and PLCA microsphere, 447,796 and U.S. Patent Application Publication No. US 2002130430), Biodegradable nanometer capsule and bioadhesive microsphere, or by protein carrier (O ' Hare and Normand, International PCT publication No. WO 00/53722).Alternatively, by direct injection or use infusion pump nucleic acid/vehicle compositions is carried out local delivery.That no matter nucleic acid molecules of the present invention carries out is subcutaneous, intramuscular still is the Intradermal direct injection, all can utilize standard pin and injecting method, or by the needleless technology, as at Conry etc., Clin Cancer Res 5:2330-2337,1999 and Barry etc., the description among the International PCT publication No. WO 99/31262.Molecule of the present invention can be used as pharmaceutics reagent.Pharmaceutics reagent can prevent, regulate its generation or treatment (relief of symptoms is preferably all symptoms to a certain degree) to curee's morbid state.

Term " part " refers to any chemical compound or molecule, and as medicine, peptide, hormone or neurotransmitter, it has with other chemical compound such as receptor is direct or the ability of non-direct interaction.Can be present in cell surface or can be intracellular receptor alternatively with the receptor of ligand interaction.Part and acceptor interaction can cause biochemical reaction, or only interact or coupling for physics.

" asymmetry hair clip " used herein means linear siNA molecule and comprises the antisense zone, the loop section that can comprise nucleotide or non-nucleotide, with justice zone is arranged, its included nucleotide lacks than antisense is regional, causes having adopted zone that enough complementary nucleotides and the regional duplex structure that matches and be formed with ring of antisense are arranged.For example, a kind of asymmetry hair clip siNA molecule of the present invention can comprise the antisense zone, its have enough in the T cell mediation RNAi length (as about 19 to the individual nucleotide in about 22 (as about 19,20,21 or 22)) and comprise that about 4 arrive the ring zones of about 8 (as about 4,5,6,7 or 8) nucleotide, with justice zone is arranged, it has about 3 to about 18 (as about 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18) nucleotide and antisense regional complementarities.Asymmetry hair clip siNA molecule also can comprise can be by 5 ' terminal phosphate group of chemical modification.The loop section of asymmetry hair clip siNA molecule can comprise that nucleotide, non-nucleotide, connexon molecule or yoke described herein close molecule.

Alleged herein " asymmetry duplex " means the siNA molecule and has two isolating chains that include justice zone and antisense zone, the nucleotide that has adopted zone to comprise herein lacks than antisense is regional, so that has adopted zone to have enough complementary nucleotides and pairing of antisense zone and formation duplex structure.For example, the double-stranded siNA molecule of a kind of asymmetry of the present invention can comprise the antisense zone, it has the enough length (arriving the individual nucleotide in about 22 (as about 19,20,21 or 22) as about 19) that mediates RNAi in the T cell, with justice zone is arranged, it has about 3 to about 18 (as about 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18) nucleotide and antisense regional complementarities.

" regulate gene expression " means and target gene expression raised or downward modulation, comprises that exist level or the mRNA of mRNA translates or target gene encoded protein or synthetic rise of protein protomer or downward modulation in the cell.The regulation and control of gene expression also can be taken place to raise or the target gene of downward modulation determines or can be the one or more albumen of this target gene coding or existence, quantity or the activity of protein protomer, so that the expression of object albumen or subunit, level or activity are higher or lower than when not having regulation and control person (as siRNA) viewed.For example, term " regulation and control " can mean " inhibition ", but the use of " regulation and control " speech is not limited thereto definition.

" inhibitions ", " downward modulation of expressing " or " minimizing " means the expression of the level of the gene of encode one or more albumen or protein protomer or RNA molecule or equivalent RNA molecule, or the one or more albumen of target gene coding or the level or the activity of protein protomer, reduce to not to be lower than when having nucleic acid molecules of the present invention (as siNA) and observe.Viewed level when the inhibition of siNA molecule in one embodiment,, downward modulation or the minimizing meaning exist for being lower than molecule deactivation or decay.Viewed level when the inhibition of siNA molecule in another embodiment,, downward modulation or the minimizing meaning exist for being lower than the siNA molecule that for example has missense (scrambled) sequence or mispairing.When in another embodiment, the inhibition of nucleic acid molecules gene expression of the present invention, downward modulation or minimizing are higher than it and do not exist.

Gene " silence " refers to make it part or function thoroughly takes place to lose by targeting inhibition of gene expression in cell, also can be described as " striking low (knock down) ".According to ambient conditions and the biological question that will illustrate, it can be preferably part and reduce gene expression.Alternatively, may expect to reduce as far as possible gene expression.Reticent degree can determine that some of them have been done summary in international publication number WO 99/32619 by the method that this area is familiar with.Rely on analytical method, gene expression quantitatively make particular of the present invention the inhibition of the various degree that may expect all can detect, comprise preventative and therapeutic method, it can strike low with mRNA level or protein level or the active expression of target gene of representing, for example be equal to or higher than 10%, 30%, 50%, 75%, 90%, 95% or 99% of foundation level (being normal level) or other control level, comprise the expression of rising that may be relevant with the particular disease states of targeted therapy or other state.

Phrase " inhibition target gene expression " refers to that siNA of the present invention starts the ability of the gene silencing of target gene.For detecting the degree of gene silencing, compare with the control sample of not expressing this construct expressing the organ of interest of particular build body or the sample or the mensuration sample of cultured cells.Control sample (not expression construct) is appointed as 100% relative value.When the value of measuring with respect to contrast is about 90%, often be 50%, and when being 25-0% in specific embodiments, promptly reached inhibition expression of target gene.Suitable detection means comprises technology for detection albumen or the mRNA level of for example utilizing known to those skilled in the art, as dot blotting, northern trace, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotype well-known to those skilled in the art detects.

" curee " means organism, tissue or cell, and it can comprise as the curee or as the organ of donor, or the receptor of transplanted cell, or the cell of the object of itself sending for siNA.Therefore " curee " can refer to organism, organ, tissue or cell, comprise external or ex-vivo organ, tissue or cell curee, nucleic acid molecule of the present invention can strengthen to its dispenser and with the polypeptide that raising polynucleotide described herein are sent sends effect.Exemplary curee comprises mammalian subject or cell, for example human patients or cell.

" cell " used herein is its common biological significance of use, and is not meant multicellular organisms integral body, for example do not refer in particular to a people.Cell can be present in the organism, as bird, plant and mammal such as people, milch cow, goat, troglodyte, monkey, pig, Canis familiaris L. and cat.Cell can be protokaryon (for example bacterial cell) or eucaryon (for example mammal or plant cell).Cell can be somatic cell or germ cell line origin, totipotency or versatility, fissionable or nondividing.Cell can be by gamete or embryo, stem cell or differentiation cell-derived or comprise these cells fully.

" carrier " means any technology based on nucleic acid and/or virus of the nucleic acid that is used to send expectation.

" comprise " and mean the content that includes but not limited to behind " comprising " speech.Therefore, the use that term " comprises " represents that listed key element is needs or essential, but other key element is optionally, may exist or not exist." by ... form " mean comprise and be limited to phrase " by ... form " after content.Therefore, phrase " by ... form " the listed key element of expression is that need or essential, and can not has other key element." basically by ... form " mean and comprise listed key element behind any this phrase, and be confined to not disturb or help in open, to refer in particular to active or other key element of acting on for listed key element.Therefore, phrase " basically by ... form " the listed key element of expression is that need or essential, but other key element is optionally, and may exist or not exist, this depends on whether they influence the active or effect of listed key element.

" RNA " means the molecule that comprises at least one ribonucleotide residue." ribonucleotide " means in 2 ' position of β-D-ribofuranose part has the nucleotide of oh group.Term " RNA " comprises double-stranded RNA, single stranded RNA, isolating RNA such as partially purified RNA, the RNA that produces of pure RNA, synthetic RNA, reorganization, and the RNA of the change different with the RNA of Lock-in by adding, lack, replace and/or change one or more nucleotide basically.This change can comprise the adding of non-nucleotide material, as is added to the terminal or inner of siNA, for example on the one or more nucleotide of RNA.The nucleotide of RNA molecule of the present invention also can comprise non-standard nucleotide, as the nucleotide of non-Lock-in or the nucleotide or the Deoxydization nucleotide of chemosynthesis.The RNA of these changes can be described as the analog of the RNA of analog or Lock-in.

" highly conserved sequence zone " means one section nucleotide sequence in the one or more zones of target gene, its significant change not in different generations or different biosystem.

" have justice zone " means one section nucleotide sequence of siNA molecule, the antisense regional complementarity of itself and this siNA molecule.In addition, the adopted zone that has of siNA molecule can comprise the nucleotide sequence that has homology with target nucleic acid sequence.

" antisense zone " means one section of the siNA molecule and has the nucleotide sequence of homology with target nucleic acid sequence.In addition, the antisense zone of siNA can randomly comprise one section nucleotide sequence that has complementarity with the adopted zone of having of siNA molecule.

" target nucleic acid " refers to express or the active any nucleotide sequence that will be regulated.Target nucleic acid can be DNA or RNA.

" complementation " refers to that nucleic acid can be by forming hydrogen bond between traditional Watson-Crick or other non-traditional type structure and another nucleotide sequence.When relating to nucleic acid molecules of the present invention, nucleic acid molecules is enough to make the correlation function of this nucleic acid to take place with the free energy that combines of its complementary series, for example the RNAi activity.Definite method in conjunction with free energy of nucleic acid molecules is known in the artly (for example to see Turner etc., CSHSymp.Quant.Biol.LII, 1987,123-133 page or leaf; Frier etc., Proc.Nat.Acad.Sci.USA 83:9373-9377,1986; Turner etc., J.Am.Chem.Soc.109:3783-3785,1987).The percentage ratio that complementary percentage ratio represents can to form between the contiguous residue of a nucleic acid molecules and second nucleotide sequence hydrogen bond (for example Watson-Crick matches) (for example, article one, there are 5,6,7,8,9 or 10 nucleotide and second to contain the nucleotide sequence pairing of 10 nucleotide in 10 of oligonucleotide nucleotide, represent 50%, 60%, 70%, 80%, 90% and 100% complementation respectively)." fully complementary " be meant a nucleotide sequence all contiguous residues can and the contiguous residue of the same quantity of second nucleotide sequence between form hydrogen bond.

Term used herein " universal base " refers to the nucleotide base analog, and it can form base pair with each n DNA/RNA base, and the repulsion between them is very little.The non-limitative example of universal base comprise C-phenyl, C-naphthyl and other aromatic derivant, inosine, methylpyrrol carboxamides and nitro-pyrrole derivant as known in the art 3-nitro-pyrrole, 4-nitroindoline, 5-nitroindoline and 6-nitroindoline (example is seen Loakes, Nucleic Acids Research 29:2437-2447,2001).

" acyclic nucleotide " used herein refers to any nucleotide with acyclic ribose sugar, and for example its any ribose carbon (C1, C2, C3, C4 or C5) is for independently or in combination lacking in nucleotide.

Term " biodegradable " used herein " refer to the degraded in biosystem, carried out, for example enzymatic degradation or chemical degradation.

Term used herein " biologically active molecules " refers to bring out in system or the chemical compound or the molecule of a kind of biologically of modification.The nonrestrictive example of biologic activity siNA molecule, be independent or with desired other molecular combinations of the present invention, comprise therapeutic activity molecule such as antibody, cholesterol, hormone, antiviral agents, peptide, protein, chemotherapeutics, micromolecule, vitamin, cofactor, ucleosides, nucleotide, oligonucleotide, enzyme nucleotide, antisensenucleic acids, triplex forming oligonucleotide, 2,5-A chimera, siNA, double-stranded RNA, allozyme, fit, bait and analog thereof.Biologically active molecules of the present invention also comprises pharmacokinetics and/or the molecule of pharmacokinetics, for example fat and polymer such as polyamines, polyamide, Polyethylene Glycol and other polyethers that can regulate and control other biological active agents.

Term used herein " phospholipid " refers to comprise the hydrophobic molecule of at least one phosphorio group.For example, phospholipid can comprise a phosphorus-containing groups and saturated or unsaturated alkyl group, randomly with OH, COOH, oxo, amine, or that replace or unsubstituted aromatic yl group replacement.

" cap sequence " means the chemical modification structure, and it is incorporated into the two ends (example is seen Adamic etc., U.S. Patent number 5,998,203, mode is by reference incorporated this paper into) of oligonucleotide herein.These terminal-modified structures can be protected nucleic acid molecules, prevent that it from being degraded by exonuclease, and help it to send and/or the localization in cell.Cap sequence can be present in 5 ' terminal (5 ' medicated cap) or 3 ' end (3 ' medicated cap) or can all exist in two ends.In non-limitative example, 5 ' medicated cap includes but not limited to glyceryl, reverse deoxidation dealkalize base residue (part); 4 ', 5 '-methylene nucleotide; 1-(β-D-erythro form furan celery sugar (erythrofuranosyl)) nucleotide, 4 '-thio nucleotides; The homocyclic nucleus thuja acid; 1,5-dewatering hexitol nucleotide; L-nucleotide; α-nucleotide; The modification nucleotide base; The phosphorodithioate connexon; Su Shi-penta furan celery ribotide; Acyclic 3 ', 4 '-disconnected nucleotide; Acyclic 3,4-dihydroxy butyl nucleotide; Acyclic 3,5-dihydroxy pentyl nucleotide; 3 '-3 '-reverse nucleotide segment; 3 '-3 '-reverse dealkalize base section; 3 '-2 '-reverse nucleotide segment; 3 '-2 '-reverse dealkalize base section; 1,4-butanediol phosphate ester; 3 '-phosphoramidate; The hexyl phosphate ester; Amino hexyl phosphate ester; 3 '-phosphate ester; 3 '-thiophosphate; Phosphorodithioate; Bridging or non-bridged methylphosphonic acid part.

The non-limitative example of 3 ' medicated cap includes but not limited to glyceryl, reverse deoxidation dealkalize base residue (part); 4 ', 5 '-methylene nucleotide; 1-(β-D-erythro form furan celery sugar (erythrofuranosyl)) nucleotide, 4 '-thio nucleotides; The homocyclic nucleus thuja acid; 5 '-amino-alkyl phosphate; 1,3-diaminourea-2-propyl phosphate; The 3-Aminopropyphosphinic acid ester; The amino hexyl phosphate ester of 6-; 1, the amino 1-isobutyl-3,5-dimethylhexylphosphoric acid of 2-; The hydroxypropyl phosphate ester; 1,5-dewatering hexitol nucleotide; L-nucleotide; α-nucleotide; The modification nucleotide base; Phosphorodithioate; Su Shi-penta furan celery ribotide; Acyclic 3 ', 4 '-disconnected nucleotide; 3,4-dihydroxy butyl nucleotide; 3,5-dihydroxy pentyl nucleotide; 5 '-5 '-reverse nucleotide segment; 5 '-5 '-reverse dealkalize base section; 5 '-phosphoramidate; 5 '-thiophosphate; 1,4-butanediol phosphate ester; 5 '-amino; Bridging or non-bridged 5 '-phosphoramidate, thiophosphate and/or phosphorodithioate, bridging or non-bridged methylphosphonic acid and 5 '-(more specific example is seen Beaucage and Lyer to the sulfydryl part, Tetrahedron 49:1925,1993; Incorporate this paper herein by reference into).

Term " non-nucleotide " refers to group or chemical compound arbitrarily, and it can be integrated into nucleic acid chains on the position of one or more nucleotide units, comprises sugar and/or phosphoric acid substituent group, and allows remaining base to show their enzymatic activity.This group or chemical compound are the dealkalize base, because it does not contain common discernible nucleotide base, therefore as adenosine, guanine, cytosine, uracil or thymus pyrimidine and 1 '-position lacks base.

Comprise natural base known in the art (standard) and modified base what this " nucleotide " of using was familiar with for this area.These bases be usually located at 1 of nucleotide sugar molecule '-position.Nucleotide generally includes base, sugar and phosphate group.This nucleotide can not modified or modified in sugar, phosphoric acid and/or base molecule position (also refers to interchangeable nucleotide as nucleic acid analog, modification, non-natural nucleotides, off-gauge nucleotide and other; See,, see before as Usman and McSwiggen; Eckstein, etc., International PCT publication No. WO 92/07065; Usman, etc., International PCT publication No. WO 93/15187; Uhlman ﹠amp; Peyman sees before, and all documents are incorporated by reference this paper at this).There is the example of many modified nucleotide bases in this area, and as at Limbach etc., Nucleic Acids Res.22:2183 sums up in 1994.Some nonrestrictive examples to the modification of base can be introduced in the nucleic acid molecules, comprise inosine, purine, pyridine-4-ketone, pyridin-2-ones, phenyl, pseudouracil, 2,4, the 6-trimethoxy-benzene, the 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkyl cytidine (as, the 5-methylcytidine), 5-alkyl uridnine (as, the ribose thymidine), 5-halogen uridnine (as, the 5-broxuridine) or 6-aza-pyrimidine or 6-alkyl pyrimidine (as, the 6-methyluracil), propinyl and other (Burgin, Deng the people, Biochemistry 35:14090,1996; Uhlman ﹠amp; Peyman sees before)." modified base " refers to 1 ' nucleotide base outside adenosine, guanine, cytosine and the uracil of position or their equivalent in this regard.

" target position " refers to a sequence in target RNA, and it is used for including in the antisense zone enzyme action of the siNA construct mediation of the sequence that is complementary to target sequence by " targeting ".

" the detectable level of enzyme action " refers to that the enzyme action (with the formation of cracked product RNA) to target RNA is enough to find the degree of enzyme action product under the background of the RNA that produces at the target RNA that degrades at random.The enzyme action product of the target RNA of 1-5% is enough to be detected under the background with most detection methods.

" living things system " refers to, test material, and under purification or non-purified form, from biogenic, including, but not limited to people, animal, plant, insecticide, antibacterial, virus or other source, wherein this system comprises the active essential composition of RNAi.Term " living things system " comprises, as, cell, tissue or organism, or its extract.The term living things system also comprises the RNAi system of the reorganization that can be used for external foundation.

At this term " biodegradable " connexon of using " refer to nucleic acid or non-nucleic acid connexon molecule; and it is designed to biodegradable connexon to connect a molecule to another molecule; for instance, and bioactive molecule is to a siNA molecule of the present invention or the having on justice and the antisense strand of siNA molecule of the present invention.This biodegradable connexon is designed so that its stability can be conditioned for specific purpose, as is delivered to specific tissue or cell type.Stable available number of chemical composition based on the biodegradable connexon molecule of nucleic acid is regulated, for instance, the nucleotide of ribonucleotide, deoxyribonucleotide and chemical modification, as 2 '-O-methyl, 2 '-fluorine-based, 2 '-amino, 2 '-O-amino, 2 '-C-pi-allyl, 2 '-O-pi-allyl and other 2 '-nucleotide modification or the base modification.This biodegradable nucleic acid connexon can be dimer, trimer, the tetramer or longer nucleic acid molecules, for instance, length of nucleotides is about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 oligonucleotide, maybe can comprise the wall scroll nucleotide that phosphate connects, for instance, phosphoramidate or di-phosphate ester.This biodegradable nucleic acid connexon molecule also can comprise nucleic acid backbone, ribose or nucleic acid base modification.

" the dealkalize base " refer to that glycan molecule lacks base or have other chemical group in the base position of 1 ' position, referring to No. the 5th, 998,203, people's such as for example Adamic United States Patent (USP).

" nucleotide of non-modification " refers to adenosine, cytosine, guanine, thymus pyrimidine or uracil, is connected on 1 ' carbon of β .-D-nuclear-furanose.

" nucleotide of modification " refers to nucleotide base arbitrarily, and it is contained in the modification on the chemical constitution of nucleotide base, sugar and/or phosphate ester of non-modification.The nonrestrictive example of the nucleotide of modification shows in formula I-VII and/or other modification described here.

As describe in the present invention with 2 '-being connected of the nucleotide of modification, refer to 2 by " amino " '-NH ₂Or 2 '-O-NH ₂, it can be modified or not be modified.Such modification group exists, and for instance, is described in people's such as people's such as Eckstein U.S. Patent number 5,672,695 and Matulic-Adamic the U.S. Patent number 6,248,878.

The siNA molecule can cooperate with cationic-liposome, by liposome, or otherwise is delivered to target cell or tissue.This nucleic acid or nucleic acid coordination compound can mix or not mix biomacromolecule, by injection, infusion pump or support topical.In another embodiment, Polyethylene Glycol (PEG) can covalently be connected to siNA chemical compound of the present invention, to improving on the polypeptide that polynucleotide send, or on both.The PEG that connects can be molecular weight arbitrarily, preferably arrives about 50,000 dalton (Da) from about 2,000.

There is adopted zone to be connected to the antisense zone, as polynucleotide connexon or non-nucleotide connexon by the connexon molecule.

" oppositely repeat " refers to comprise that tool orientates as when the nucleotide sequence that adopted and antisense composition are arranged that repeats can form when being transcribed double-stranded siRNA.Oppositely repeat optional connexon or the heterologous sequence of comprising, as between multiple two compositions from nickase.Reverse multiple composition has the length that is enough to form double-stranded RNA.Typically, oppositely multiple each composition length is about 15 to about 100 nucleotide, preferred about 20-30 nucleotide base, the preferred about 20-25 of a length nucleotide is as 20,21,22,23,24,25,26,27,28,29 or 30 length of nucleotides.

" nucleic acid " refers to deoxyribonucleotide or ribonucleotide and its polymer of list or double chain form.This term comprises this nucleic acid, and it comprises known nucleotide analog, and the framework residue of modification or bonding for synthetic, abiogenous and non-abiogenous, have similar binding characteristic to reference nucleic acid, with the mode metabolism similar in appearance to reference nucleotide.The example of these analog comprises that tool is not restricted, thiophosphate, phosphamide ester, methyl phosphorodithioate, chirality-methyl phosphorodithioate, 2-O-methyl ribonucleotides, peptide nucleic acid(PNA) (PNA).

" big double-stranded RNA " refers to have arbitrarily the double-stranded RNA greater than about 40 base pairs (bp) size, for instance, and greater than 100bp or more especially greater than 300bp's.The sequence of big dsRNA can be fragment or the whole mRNA of mRNA.The size of the maximum of big dsRNA does not have restricted at this.This double-stranded RNA can comprise the base of modification, can be to phosphoric acid sugar skeleton or to the nucleoside modification in this position.Such modification can comprise nitrogen or sulfur heteroatom or other modification known in the art arbitrarily.

This duplex structure can be by self complementary RNA chain formation, as passing through hair clip or microRNA or forming by two different complementary RNA chains are annealed.

" overlapping " refers to that two RNA fragments have the eclipsed sequence of a plurality of nucleotide on a chain, and this a plurality of nucleotide (nt) number can lack as a 2-5 nucleotide or a tool 5-10 nucleotide or more.

" one or more dsRNA " refers to based on sequence, mutual different dsRNA.

" target gene or mRNA " refers to any interested gene or mRNA.In fact arbitrarily the gene that passes through heredity or sequence analysis identification before can be target.Target gene or mRNA can comprise development gene and controlling gene, also can be gene metabolic or structural gene or codase.Target gene can be expressed in observing those cells of phenotype or expressed in organism in the direct or indirect mode that influences the phenotype sign.Target gene can be endogenous or external source.These cells comprise adult or embryo animal or contain the intravital cell arbitrarily of plant of gamete or arbitrarily as the isolated cells that produces in immortal cell line or primary cultured cell.

In this description and the claims of enclosing, singulative " a ", " an " and " the " are unless contain multiple indication clearly qualification in addition in the text.

In this certain embodiments fully for illustration and be not meant to restriction as the scope of the present invention in claims, described.Though used special term and numerical value at this, these terms and numerical value can be made example by understanding and be not used in to limit the scope of the invention.

The all publications in the disclosure, quoted and reference material for all purposes at this complete by reference this paper that incorporates into.

Embodiment

The present invention has been described in top disclosing substantially, and it is by the further example of the following example quilt.These embodiment that describe are for illustration fully, and are not meant to and limit the scope of the invention.Though used special term and numerical value at this, these terms and numerical value are interpreted as exemplary and are nonrestrictive to the scope of the invention.

Embodiment 1

The testing program and the method for utilization

Synthesizing of siRNA/ polypeptide conjugates

Polypeptide and RNA use the solid-phase synthesis preparation of standard.Polypeptide and RNA molecule must make their covalency interconnect with specific molecular, thus functionalization.For polypeptide, N-terminal 3-maleimide propanoic acid functionalization.Yet we recognize that other functional group such as bromine or iodine acetyl moiety also can work.To 5 of 5 of RNA molecule sense strand ' end or antisense strand ' end 1-O-dimethoxytrityl-hexyl-disulphide connexon functionalization.

The RNA oligomer is dissolved in the 0.5mL water that is added with 2ml buffer A (20mM Tris-HCl, pH6.8,50% Methanamide).Then add polypeptide PN277, it causes precipitation to form, and adds the 2M TEAA buffer postprecipitation dissolving of 1mL.After reaction was finished, decompression went down to desolventize, and with the solid dissolving in buffer A (20mM Tris-HCl, pH6.8,50% Methanamide) that generates.Material is loaded on the Amersham Resource Q post and with the buffer A flushing with 5 times of column volumes in 6mL/ minute.Finish separation with the buffer B (20mM Tris-HCl, pH6.8,50% Methanamide, 1M NaCl) of 15 times of column volumes from the operation of 15-60% linear gradient with 6mL/ minute flow velocity.The conjugates of purification blocks (3.5K MWCO) PBS desalination relatively with the slidalyzer dialysis.Measure the amount of conjugates with spectrophotometer method based on the molar absorption coefficient that calculates when the λ=260nm.With RP-HPLC the analysis of the purity of this conjugates is shown in the back.

In the HEPES pH7.4 of the magnesium acetate of the potassium acetate of 50mM, 1mM and 15mM, 90 ℃ of heating after 2 minutes in 37 ℃ of incubations 1 hour, the antisense RNA chain that conjugates to polypeptide is annealed to its complementary sense RNA chain.Formation with non-degeneration (15%) polyacrylamide gel electrophoresis and ethidium bromide staining conclusive evidence double-stranded RNA conjugates.

The example of a siRNA/ polypeptide conjugates of the present invention shows in embodiment 3.

Array structure under the conjugates tool of 5 of the preparation 1 of example conjugates: peptide PN277 (H2B 13-48) (SEQ ID NO:37) and antisense strand CN950asen (N163asen) (SEQ ID NO:60) ':

Oligomerization CN950asen is dissolved in the 0.5mL water of the buffer A (20mM Tris-HCl, pH6.8,50% Methanamide) that is added with 2mL.Add peptide (PN0277) then, it causes precipitation to form, and is dissolved at the TEAA buffer postprecipitation that adds 1mL 2M.After reaction was finished, decompression was gone down to desolventize and the solid that produces is dissolved (20mM Tris-HCl, pH6.8,50% Methanamide) in buffer A.Material is loaded on the Amersham Resource Q post and with the buffer A flushing with 5 times of column volumes in 6mL/ minute.Finish separation with the buffer B (20mM Tris-HCl, pH6.8,50% Methanamide, 1M NaCl) of 15 times of column volumes from the operation of 15-60% linear gradient by flow velocity with 6mL/ minute.With the conjugates desalination of the relative PBS of slidalyzer dialysis card (3.5K MWCO) to purification.Measure the amount of conjugates with spectrophotometer method based on the molar absorption coefficient that calculates when the λ=260nm.Determine the purity of this conjugates with RP-HPLC.By 90 ℃ of heating after 2 minutes in 37 ℃ of incubations 1 hour, peptide conjugates antisense strand and complementary antisense strand are annealed in the HEPES pH7.4 of the magnesium acetate of the potassium acetate of 50mM, 1mM and 15mM.Formation with non-degeneration (15%) polyacrylamide gel electrophoresis and ethidium bromide staining conclusive evidence double-stranded RNA conjugates.

The preparation 2 of example conjugates: synthesize following conjugates with method of describing in the preparation 1 of above example conjugates and program:

The conjugates of peptide PN277 (SEQ ID NO:37) and oligomerization CN952sen (N163sen) (SEQ ID NO:59) has following array structure:

The Dicer substrate 27-mer conjugates of peptide PN277 and oligomerization CN740sen (SEQ ID NO:63) has following array structure:

The Dicer substrate 29-mer conjugates of peptide PN277 and oligomerization CN741asen (SEQ ID NO:64) has following array structure:

Cell and cell culture

The mouse tail fibroblast is derived from the tail of C57BL/6J mice or tg197 hTNF-α transgenic mice.Take off tail and be cut into segment with blade.Clean segment 3 times with PBS, use then 0.5mg/ml collagenase, 100 units/ml penicillin and 100 μ g/ml streptomycins in shaking table in 37 ℃ of incubations with meristem.In DMEM culture medium, cultivate the tail segment then with fill-in (as described above).The 9L/lacZ cell of the constitutive expression LacZ that obtains from ATCC (#CRL-2200) is a rat glioma sarcomatosum fibroblast, also grows in the DMEM culture medium with fill-in (as above describing).

In the Dulbecco culture medium (IMDM) of the Iscove improvement that contains 4mM L-glutamate, Glu, non essential amino acid and 10% hyclone, keep isolating human monocyte.Mouse tail fibroblast (MTF) is kept in the dulbecco minimum essential medium Dulbecco (DMEM) of the Dulbecco of the fill-in that contains 1mM Sodium Pyruvate, non essential amino acid and 20% hyclone improvement.All cells are in 37 ℃ and 5%CO ₂In cultivate, be supplemented with and contain 100 units/ml penicillin, 100 μ g/ml streptomycins and 0.25mg/ml amphotericin B (Invitrogen Carlsbad, antibiotic cocktail CA).

Human monocyte's separation and purification

(Temecula CA) buys from Golden West Biologicals from healthy supplier's fresh human blood sample.Be used for separating monocytic cell receiving that the back is diluted with PBS blood sample at once with 1: 1 ratio.(NJ) gradient method is from separation of whole blood for Amersham, Piscataway by Ficoll at first for peripheral blood lymphocytes (PBMC).Further use the positive selective reagent box of Miltenyi CD14 (MILTENYI BIOTEC GmbH, Germany) to be purified into mononuclear cell from PBMC according to manufacturer specification.(BD Bioscience, San Jose CA) behind the staining cell, learn that with the flow cytometry evaluation purity of mononuclear cell goods is higher than 95% using anti-CD14 antibody.Before inducing and striking harmonic analysis, the human monocyte of purification (above-described) in complete medium keeps and spends the night.

Human monocyte's activation

(Sigma, St Louis MO) carry out human monocyte's activation to cell culture moderate stimulation tumor necrosis factor (TNF-α) generation to lipopolysaccharide LPS by adding 0.1-1.0ng/ml.With LPS incubation collecting cell and analyze by Quantigene according to the description of manufacturer that (Genospectra, Fremnont CA) measure the level of mRNA after 3 hours.According to the testing program of manufacturer, (BD Biosciences, San Jose CA) measure the variation of inducing back TNF-alpha levels with ELISA.

Human TNF-α strikes harmonic analysis mRNA (bDNA) and protein (ELISA)

Mononuclear cell is inoculated in the OptiMEM in a tool 100K cell/100 μ l/ holes, and (96 orifice plates USA) strike low test to carry out TNF-α for Invitrogen, Carlsbad.Peptide and siRNA were mixed with OptiMEM 5 minutes, add hyclone (FBS) ultimate density to 3%FBS in the mixture then.According to the testing program of manufacturer, with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) transfectional cell.All transfections are passed through cell in 37 ℃ and 5%CO ₂Incubation 3 hours removes transfection reagent then, replenishes complete medium and gives cell and overnight incubation and carry out.Carry out bDNA according to the description of manufacturer and analyze (from Genospectra, Fremont, the Quantigene analysis of CA).Sample from cell culture medium is directly used in elisa assay.The plasma sample that from the separation of whole blood of collecting by the eye socket blood-letting is used to comprise the experimental analysis of transgenic mice.Measure the level (R﹠amp of blood plasma hTNF-α with ELISA with 1: 2 dilution according to the description of manufacturer; D system, Minneapolis, MN).

Fluidic cell detects

(Fullerton CA) carries out flow cytometric analysis with Beckman Coulter FC500 cytoanalyze.Fluorescent probe (FAM or Cy5 are used for siRNA and FITC or PE and are used for CD14) according to utilization rectifies an instrument.Propidium iodide (Fluka, St Louis, MO) and annexin V (BD Bioscience, San Jose is CA) as cell survival and Cytotoxic index.

The siRNA degradation analysis of Dicer mediation

Whether siRNA and siRNA/ polypeptide conjugates and Dicer Cobra venom endonuclease (Stratagene Cat.#240100) incubation easily is degraded to detect them.Scheme according to manufacturer is carried out.In brief, digestion reaction carries out in 10 μ L cumulative volumes, and allows to be incubated overnight in 37 ℃.Spend the night behind the incubation, the sample of 2 μ L of digestion mixture mixes with the last sample dyestuff of 2X, and analyzes by the gel electrophoresis on the 15%TBE of the polyacrylamide gel that contains carbamide and the non-degeneration of 15%TBE.

With the degraded of LC-MS monitoring Dicer to RNA

The LC-MS service condition is that temperature control is at 65 ℃ with XTerra C18 post, 2.5 μ m, 2.1 * 50mm (Waters company).Mobile phase is 100mM hexafluoroisopropanol, 7mM triethylamine and 100% methanol eluant.Gradient is 5-16% in 40 minutes processes.Eluant divides among the inflow PDA and WatersMicromass ZQ ESI list-quad mass spectrograph moves under the anion pattern.Capillary voltage is that 3.0kV and taper hole voltage are 45V.Desolventizing carries out at 300 ℃, is aided with 600L/ hour N ₂The source is controlled at 90 ℃.The sampled scan rate be 1 second 1000-2000m/z.

Embodiment 2

Screening tool hTNF-α strikes SA Dicer endonuclease zymolyte SiRNA

The present embodiment illustration exemplary siRNA of the present invention, its screened so that hTNF-α gene expression dose effectively reduces.The importance of aiming hTNF-α gene is that this gene is with the generation of regulating rheumatoid arthritis (RA) or develop relevant because when overexpression in human or other mammalian subject.Therefore, the reduction hTNF-α gene expression of targeting can be used for treating RA.

Recent evidence shows the long RNA duplex of 25-30 nucleotide that is, with respect to short RNA duplex, can reach the effect of the reduction expression of target gene level that surpasses 100 times.This enhanced low activity that strikes is the substrate of dicer Cobra venom endonuclease (RNAse III) owing to these long siRNA duplexs.Following table 3 shows that siRNA duplex length range is 21 to 27 nucleotide.YC12 and N161-N164 have been selected by sequence specific post transcriptional gene silencing hTNF-α GeneScreen.Initial screening process comprises (Invitrogen, Carlsbad, Ca) the transfection human monocyte of the stimulation in the independent siRNA of table 3 to LPS that tabulates with Lipofectamine 2000.

Table 3

Targeting is in the siRNA of hTNF-α

siRNA	Nucleotide sequence
		YC12	SEQ ID NO 53 5′-GCCUGUACCUCAUCUACUCUU-3′ SEQ ID NO 54 3′-UUCGGACAUGGAGUAGAUGAG-5′
N161	SEQ ID NO 55 5′-GCCUCUUCUCCUUCCUGAUCGUGdGdC-3′ SEQ ID NO 56 3′-GUCGGAGAAGAGGAAGGACUAGCACCG-5′
		N162	SEQ ID NO 57 5′-GCCUGCUGCACUUUGGAGUGAUCdGdG-3′ SEQ ID NO 58 3′-GACGGACGACGUGAAACCUCACUAGCC-5′
N163	SEQ ID NO 59 5′-CCCAUGUGCUCCUCACCCACACCdAT-3′ SEQ ID NO 60 3′-GUGGGUACACGAGGAGUGGGUGUGGUA-5′
		N164	SEQ ID NO 61 5′-ACCUCAUCUACUCCCAGGUCCUCdTdT-3′ SEQ ID NO 62 3′-CAUGGAGUAGAUGAGGGUCCAGGAGAA-5′

CN740 has justice (5 '-3 ')	SEQ ID NO 63 5′-GCCUGUACCUCAUCUACUCCCAGGUCC-3′
		CN741 antisense (5 '-3 ')	SEQ ID NO 64 5′-GGUCCUGGGAGUAGAUGAGGUACAGGCUU-3′

The sequence of YC12 and N161-N164 with 5 '-3 ' have justice (on) and the mode of 3 '-5 ' antisense (descending) in table 3, list.

(Invitrogen Carlsbad is Ca) with among 0.16nM, 0.8nM, 4nM and 20nM concentration transfection YC12 and the N161-N164 each with Lipofectamine 2000.The TNF-α that table 4 has been summed up the every kind of siRNA that represents with percentage rate strikes low activity.Qneg representative at random the siRNA sequence and as negative control.Observed Qneg strikes the low activity that strikes that low activity is standardized as 100% (100% gene expression dose) and every kind of siRNA and represents in the mode with respect to negative control.Data show in the table 4 is compared with Qneg siRNA negative control, and siRNA N161, N162 and N164 have no significant effect the level of TNF-α mRNA.By contrast, siRNAY12 reduces the Qneg negative control level of the level to 66% of TNF-α mRNA, and siRNA N163 reduces the Qneg negative control level of the level to 57% of TNF-α mRNA.

Data show dicer substrate siRNAN163 in the table 4 and YC12 have reduced the level of hTNF-α mRNA among the human monocyte effectively.Selected siRNAN163 to be used for further sign.

Table 4

The TNF-α of the siRNA of liposome transfection strikes low activity

Embodiment 3

SiRNA/ polypeptide conjugates reduces effectively

The level of hTNF-α mRNA in the human monocyte

Present embodiment proves, and is fashionable when the polypeptide yoke that dicer substrate siRNA of the present invention and exemplary raising polynucleotide of the present invention are sent, and reduces hTNF-α mRNA expression effectively.Summing-up has shown the low activity that strikes of siRNA/ polypeptide conjugates in following table 5.

Embodiment 2 invading the exterior 4 data presented show when using Lipofectamine 2000 (Invitrogen, Carlsbad, Ca) during transfection, siRNA N163 has effectively reduced hTNF-α mRNA level, and therefore siRNAN163 is the outstanding candidate that is used for the treatment of and/or prevents the relevant inflammatory states of one or more TNF-α.Yet cationic-liposome can be had a cytotoxicity, therefore when the treatment disease, is not suitable for sending in the body utilization.Thus, for overcoming the cationic-liposome-mediated defective of sending, exemplary N163 siRNA of the present invention conjugated to improve on the polypeptide that polynucleotide send, when as delivery vehicle, this conjugates has minimum to there not being cytotoxicity.Conjugate on the dicer substrate form (27-mer) of siRNA sequence and can before RISC loads, make polypeptide sending polypeptide, reduced polypeptide like this and can suppress the active probability of RISC from the siRNA removal.In fact, these conjugatess are sent precursor siRNA effectively to Cytoplasm and processing in a single day, the mediation of just effectively striking the target gene of low expectation.

The polypeptide (PN277) that exemplary raising polynucleotide of the present invention are sent is derived from the proteinic aminoacid sequence of human histone 2B (H2B), and its primary structure is as follows:

PN277

NH2-KGSKKAVTKAQKKDGKKRKRSRKEFYSVYVYKVLKQ-amide (SEQ ID NO:37)

The generation of conjugates is that the polypeptide PN277 that sends by the exemplary raising polynucleotide of covalently bound the present invention is to 5 ' end of the sense strand of exemplary N163siRNA of the present invention or 5 ' end of antisense strand.The example of siRNA/ polypeptide conjugates is as follows:

Polypeptide PN277 conjugation is to N163 siRNA (dsCoP277nfR950; SEQ ID NO:37 and 35) 5 of antisense strand ' end.The sense strand of N163 siRNA does not show.

Polypeptide PN277 conjugates to N163 siRNA (dsCoP277nfR952; SEQ ID NO:37 and 36) 5 of sense strand ' end.The antisense strand of N163 siRNA does not show.

Conjugate at this polypeptide PN277 and do not show as 5 ' end of the sense strand of the Qneg siRNA of negative control.

In the present embodiment, each above-mentioned siRNA/ polypeptide conjugates comprises Qneg siRNA/ polypeptide conjugates, with concentration and the human monocyte's incubation of 1nM, 10nM, 100nM and 200nM.The TNF-α that table 5 has gathered every kind of siRNA/ polypeptide conjugates representing with percentage rate strikes low activity.Observed Qneg is struck low activity be standardized as 100% (100% gene expression dose).And the low activity that strikes of every kind of siRNA is represented with the percentage rate of relative negative control.

Whether covalently bound data in the table 5 show that N163 siRNA duplex conjugates to and improve the expression that polypeptide that polynucleotide send has reduced hTNF-α mRNA effectively, do not consider 5 ' end of the polypeptide sense strand to N163 siRNA molecule or 5 ' end of antisense strand.More specifically, siRNA/ polypeptide conjugates dsCoP277nfR952 reduces hTNF-α mRNA level to 62% of Qneg/ polypeptide conjugates negative control mRNA level, and dsCoP277nfR950 reduces hTNF-α mRNA level to 38% of Qneg/ polypeptide conjugates negative control mRNA level.

Table 5

SiRNA Dicer substrate polypeptide conjugates TNF-α strikes low activity

Data presented has disclosed this surprising and unexpected discovery in the table 5, and when conjugating to the polypeptide that exemplary raising polynucleotide of the present invention send, exemplary dicer substrate siRNA of the present invention has reduced hTNF-α mRNA expression effectively.And conjugates strikes the low activity that strikes that low activity has surpassed the identical siRNA that sends by lipofectamine.

Embodiment 4

Conjugate to and improve polynucleotide and send polypeptide

SiRNA is processed by the Dicer Cobra venom endonuclease

This embodiment confirms that dicer Cobra venom endonuclease (RNase III) degraded conjugates to the siRNA that the raising polynucleotide are sent polypeptide.In the present embodiment, the three kinds of siRNA/ polypeptide conjugatess (Qneg, dsCoP277nfR950 and dsCoP277nfR952) that in embodiment 3, show incubation when existing or not having the dicer Cobra venom endonuclease.In addition, tool does not improve N163siRNA duplex incubation when existing or not having the Dicer Cobra venom endonuclease that polynucleotide are sent polypeptide.Purpose is whether detect siRNA be whether the target and the polypeptide of the degraded of dicer mediation is removed from covalently bound siRNA duplex, and it is very important in the inhibition that siRNARISC is loaded that stops the polypeptide mediation.The PTGS that may stop target gene to the inhibition of complex RISC.

Contrast by polyacrylamide gel electrophoresis shows in Fig. 1.In Fig. 1, Qneg siRNA/ polypeptide conjugates non-dicer incubation and the dicer incubation, as anticipation, all move single band into the same molecular amount, show that QnegsiRNA/ polypeptide conjugates is not degraded when existing or not having the dicer Cobra venom endonuclease.On polyacrylamide gel, when not having the dicer Cobra venom endonuclease, exemplary N163 siRNA of the present invention (non-polypeptide) migration is single band.Yet, when itself and dicer Cobra venom endonuclease incubation, observed short slightly N163 siRNA duplex, be the band that is in a ratio of a little little molecular weight with the N163 siRNA of incubation not according to evidence.Passing through 5 of antisense strand (dsCoP277nfR950) ' end, covalently bound to the exemplary N163 siRNA that improves the polypeptide that polynucleotide send with by 5 of sense strand (dsCoP277nfR952) ' end, covalently bound between the N163 siRNA that improves the polypeptide that polynucleotide send, observe the degraded of diversity.Two kinds of siRNA/ polypeptide conjugatess all as expecting like that, move the tangible band that does not have the equimolecular quantity of degrading for showing when not having dicer.Yet, when having the dicer Cobra venom endonuclease, observe the band of two kinds of different molecular weights of dsCoP277nfR952 (sense strand bonding).The molecular weight of first band equates with undegradable dsCoP277nfR952 and the second tool is equal to the molecular weight of the N163 siRNA (non-polypeptide) that dicer degraded, and it shows that covalently bound N163 siRNA separates from polypeptide.(that is, undegradable N163 siRNA/ polypeptide) density is higher than about 2 times of low-molecular-weight band (that is, the N163 siRNA/ polypeptide of degraded) to high-molecular weight band, and it shows that most of dsCoP277nfR952 (sense strand bonding) does not degrade.By contrast, dsCoP277nfR950 (antisense strand bonding) shows the high character that is subject to the degraded of dicer mediation, and institute be the equal-sized band of the N163 siRNA (non-polypeptide) that degrades of the band that equates with undegradable dsCoP277nfR950 of the size a little less than relatively and stronger molecular weight and dicer according to evidence.Compare dsCoP277nfR952 (sense strand bonding), the level of the degraded that is subject to the dicer mediation that siRNA/ polypeptide dsCoP277nfR950 (antisense strand bonding) tool is high relatively is with dsCoP277nfR950 (antisense strand bonding; Reference example 2) the ability height correlation is hanged down in observed higher striking in.Because higher siRNA/ polypeptide dicer susceptibility indicating the polypeptide mediation that reduces to complex RISC interference performance, and therefore indicate higher silence to target gene.

These data show that the exemplary siRNA that conjugates to the polypeptide that raising polynucleotide of the present invention send is subject to the degraded of dicer mediation, and represent ideal purpose for reticent target gene to be used for the candidate of effective delivery treatments precursor siRNA to cell.

Dicer endonuclease enzymatic degradation siRNA N163 produces 21-met RNA.Fig. 2 shows that non-yoke closes the RP-HPLC analysis of dicer Cobra venom endonuclease processing dynamics of siRNA N163 duplex.The RP-HPLC that in Fig. 2 (A), shows unprocessed N163 duplex.In Fig. 2 (B-E), show and dicer Cobra venom endonuclease incubation (B) 1 hour, (C) 2.5 hours, (D) 5 hours and (E) 7 hours the RP-HPLC of N163 duplex.These data show in the drawing of Fig. 3.

Analyze the feature of having determined as demonstration in Fig. 2 (E) of passing through the RNA of dicer endonuclease enzymic digestion siRNA N163 after 7 hours by ESI-MS, as in Fig. 4, showing.Fig. 4 shows being the peak at 6606.6 places and being the peak at 6965.7 places corresponding to the 21-mer antisense strand dicer pyrolysis product of N163 in quality in quality corresponding to the 21-mer sense strand dicer pyrolysis product of N163.

Determined the feature of the RNA behind the siRNA N163 that dicer endonuclease enzymatic degradation yoke closes by the ESI-MS analytic process, as shown in Figure 5.Shown that in Fig. 5 the dicer Cobra venom endonuclease adds the ESI-MS that yoke that instrument conjugates to the polypeptide PN857 of siRNA N163 closes siRNA.Aminoacid sequence and its main structure that the polypeptide PN857 that exemplary raising polynucleotide of the present invention are sent comes from human histone 2B (H2B) are as follows:

PN857

Mal-KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO:52)

Polypeptide PN857 conjugates to 5 ' end of the antisense strand of N163siRNA.

In Fig. 5 (A), obtained not exist the contrast incubation result of the conjugates duplex of dicer Cobra venom endonuclease.Fig. 5 (A) has shown that the quality corresponding to the conjugates of the antisense strand of the 27-nt of N163 and polypeptide PN857 is 13436.2 peak and is 7835.3 peak corresponding to the quality of the sense strand of the 25-nt of N163.

In Fig. 5 (B), when having obtained to exist the dicer Cobra venom endonuclease, with 8 hours result of conjugates duplex incubation.Fig. 5 (B) display quality be 13436.1 corresponding to the 27-nt antisense strand of N163 and the conjugates of polypeptide PN857, quality is 7835.6 the sense strand corresponding to the 25-nt of N163, quality is that the 21-mer antisense strand dicer pyrolysis product of 6966.3 the N16327-mer that closes corresponding to yoke and quality are 6607.6 the peak corresponding to the 21-mer sense strand dicer pyrolysis product of N163 25-mer.

Embodiment 5

Dicer substrate siRNA/ polypeptide conjugates does not cause ifn response

Present embodiment has confirmed that when with human monocyte's incubation, exemplary siRNA/ polypeptide conjugates of the present invention does not cause ifn response.The reactivity of interferon is the potential untoward reaction with the siRNA transfectional cell.Therefore, carried out in vitro study and whether caused ifn response to estimate siRNA/ polypeptide dsCoP277nfR950 (antisense strand bonding) and dsCoP277nfR952 (sense strand bonding).Qneg siRNA/ polypeptide conjugates (CoP840) is as negative control, and IF1 (IFN-1) is as positive control.Every kind of conjugates of concentration determination with 1nM, 10nM, 100nM and 200nM.Analyze ifn response with molecular marker MIP1 α.Such as expected, Qneg siRNA/ polypeptide conjugates is not induced the MIP1 alpha expression, and the inductive MIP1 alpha expression of positive control IFN-1 amount is remarkable.Conjugates dsCoP277nfR950 induces the MIP1 alpha levels similar to Qneg siRNA/ polypeptide negative control with dsCoP277nfR952, show showing effective TNF-α and strike in the SA concentration, these conjugatess do not cause ifn response (reference table 5, embodiment 3).

Embodiment 6

Transfection Vero cell on 24 orifice plates

Present embodiment openly guides siRNA to arrive the method for target cell.

The Vero cell is grown in the DMEM culture medium that contains 10%FBS, 2mL L-glutamic acid, 10nM Hepes, 100 units/ml penicillin/streptomycin.Transfection the previous day, the Vero cell that logarithmic (log) phase is cultivated is inoculated in 24 orifice plates with 50,000 cells/well.Before the transfection, for all siRNA dosage is ready to suitable L2K (Lipofectamine ^TM2000; Invitrogen) stock solution.With L2K progressively be mixed into OptiMEM

Cell culture medium (Invitrogen) and being added among the siRNA of dilution.Incubation mixture 15-30 minute is to form the transfection complex under room temperature.After incubation finished, sucking-off culture supernatants and the 150 μ l DMEM culture medium that will contain 10%FBS added in each hole.Then each transfection composite with 150 μ l adds in each hole in triplicate.Waggle gently is with the Mixed culture plate.After 3 hours, remove adding 1ml complete medium in supernatant and the every hole in 37 ℃ of incubations.The incubation cell spend the night and with microscopic examination fluorescence so that survey Cy5-siRNA.Second day, add 1.5 μ l Hoescht stock solutions by every hole and dye so that survey toxicity with Hoescht DNA staining pair cell.Rotate orifice plate gently and in 37 ℃ of incubations 15 minutes.Detect cell by Cy5 and UV filter lens with fluorescence microscope immediately.With differing the toxicity that detects the transfection composite pair cell.

Embodiment 7

Hemagglutinin is analyzed (HA analysis)

Present embodiment discloses the hemagglutinin analysis and can be used for calculating from having infected the titre of the virus that viral cell produces.

Hemagglutinin is analyzed the virus that (HA) is used for quantitative cell conditioned medium liquid.Some Viraceaes as cold virus, have surface or envelope protein, can condense (adhesion) mankind or animal erythrocyte (RBC) and at RBC surface combination N-n acetylneuraminic acid n.Because every kind of virus has many cell surface proteins, a kind of virus can be sticked more than a kind of RBC.The result of this situation can be caused forming a kind of RBC " screen work (lattice) " type by viral interconnection in conjunction with viral RBC.In case screen work has formed, can calculate the RBC of cohesion and can measure virus titer.With respect to the virus titer of not using in the siRNA cells transfected, transfection the titre of the virus in the cell of siRNA descend, show that the siRNA molecule has disturbed the one or more expression of gene of target viral.

The PBS solution for preparing 0.5% chicken red blood cell, every 20ml are used for 4 plates.Shift in each hole of 100 μ l from first row of the culture supernatant of each sample 96 hole flat boards at the bottom of the v shape.Change suction nozzle when handling different sample.If aseptic requirement is arranged to be operated in incubator for tissue culture.Use the multiple tracks adjustable pipette, every hole adds 50 μ l PBS in except other hole of the flat board of first row that obtains viral supernatant.From first row, draw 50 μ l culture supernatant and mix with the multiple tracks adjustable pipette with 50 μ l PBS of second row.Draw up and down 3 times with pipet.Mix from the sample of the dilution of the second row absorption, 50 μ l and with the 50 μ lPBS of the third line with the multichannel pipettor.Draw up and down 3 times with pipet.Repeat 4 to 5 times up to arriving last column.Remove the dilute sample of 50 μ l.Add 50 μ l, 0.5% chicken red blood cell in each hole.Dull and stereotyped 1 hour of incubation on ice.Read HA result and calculate virus titer.

Embodiment 8

24-hole Vero cell transfecting/infection titer strikes low screening and analyzes

Can present embodiment disclose and can be used for measuring that otch arranged or duplex siRNA jaggy of the present invention and reduce one or more viral target gene expression.

In this embodiment, mammiferous cell is with otch or the duplex siRNA transfection that comes from viral RNA together jaggy are arranged.After the transfection, cells transfected is with containing one or more sequence homologies in the viral infection according to the gene of the siRNA molecule of transfection of the present invention.

Method usefulness according to embodiment 6 has otch or duplex siRNA transfection Vero cell jaggy.When siRNA and Vero cell incubation finish soon, for the transfection step prepares viral dilution liquid.The virus dilution sample is so that reach the infection multiplicity (MOI) of expectation.Virus sample is diluted in PBS/BSA/PS, and the viral solution of appropriate amount is joined in each hole., after 1 hour a certain amount of transfection media (contain 0.3%BSA, 2mL L-glutamic acid, 10nM Hepes and 100 units/ml penicillin/streptomycin and add tryptic DMEM culture medium) is joined in each hole in 37 ℃ of incubations.In 37 ℃ of incubations 48 hours.After incubation finishes, collect supernatant and in 4 ℃ of storages.Disturb by the viral RNA that carries out HA analysis to measure siRNA guiding according to embodiment 7.

Embodiment 9

The screening tool strikes SA

Influenza specificity dicer endonuclease zymolyte siRNA

Present embodiment illustrates the exemplary influenza specific siRNA that is presented on this, screened come out because it can effectively reduce influenza virus titre in the Vero cell of infection.With respect to the virus titer in the non-targeting contrast siRNA cells transfected, be that it shows that the specific siRNA molecule of influenza has disturbed the viral one or more expression of gene of target with the importance of the reduction of virus titer in the cell of influenza specificity transfection siRNA.

Below table 6 show the siRNA duplex special to influenza, its with 5 '-3 ' have justice (on) and 5 '-3 ' antisense (descending) mode list.

Table 6

Influenza specificity Dicer substrate siRNA

27mer DX#	25mer matches body	The siRNA sequence
			DX2852	G3817 (DX2825)	SEQ ID NO:65 have justice 5 '-AGACAGCGACCAAAAGAAUUCGGdAdU-3 ' SEQ ID NO:66 antisense 5 '-AUCCGAAUUCUUUUGGUCGCUGUCUdTdT-3 '
DX2855	G6124 (DX2820)	SEQ ID NO:67 have justice 5 '-AUGAAGAUCUGUUCCACCAUUGAdAdG-3 ' SEQ ID NO:68 antisense 5 '-CUUCAAUGGUGGAACAGAUCUUCAUdTdT-3 '
			DX2858	G6129 (DX2819)	SEQ ID NO:69 have justice 5 '-GAUCUGUUCCACCAUUGAAGAACdUdC-3 ' SEQ ID NO:70

		Antisense 5 '-GAGUUCUUCAAUGGUGGAACAGAUCdTdT-3 '
			DX2861	G8286 (DX2822)	SEQ ID NO:71 have justice 5 '-UUGAGGAGUGCCUGAUUAAUGAUdCdC-3 ' SEQ ID NO:72 antisense 5 '-GGAUCAUUAAUCAGGCACUCCUCAAdTdT-3 '
DX2956	G1498 (DX2744)	SEQ ID NO:73 have justice 5 '-GGAUCUUAUUUCUUCGGAGACAAdTdG-3 ' SEQ ID NO:74 antisense 5 '-CAUUGUCUCCGAAGAAAUAAGAUCCdTdT-3 '

Transfection the previous day is in the 100 μ l 10%FBS/DMEM culture medium of every hole, with 1.5 * 10 ⁴The every hole inoculation of individual cell Vero cell is to 96 hole flat boards.100,20 or every kind of influenza specific siRNA of 5nM or non-targeting contrast siRNA Qneg mixes with the 0.3 μ l (1mg/mL stock solution) of Lipofectamine 2000 (Invitrogen) and at room temperature in (Gibo) incubation 20 minutes of the OptiMEM of 25 μ l (cumulative volume).Remove the supernatant in every hole of the Vero cell of inoculating, and add 75 μ l 10%DMEM complete mediums.Be incorporated in 25 μ l siNA/Lipofectamine mixture among the OptiMEM then in every hole.Each hole test is three times under every kind of condition.Prepared and do not had an other control wells of transfection conditions.After the transfection 3 hours, remove culture medium.With 100 μ l, the 1 * PBS that contains 0.3%BSA/10mM HEPES/PS clean every hole 1 *.The PR8 influenza virus that adds 30 μ l with MOI=0.1 is used for infecting in every hole.At room temperature swing plate is 1 hour.Because cell begins to separate after the transfection, so contain the tryptic 100 μ l DMEM of 0.3%BSA/10mM HEPES/PS+4 μ g/ml careful the adding to each hole from the limit, hole.Dull and stereotyped in 37 ℃, 5%CO2 incubation 48 hours.50 μ l supernatant in every hole carry out twice test by the HA analytic process.

Among DX2852, DX2855, DX2858, DX2861, DX2956 and G1498 influenza specificity dicer substrate siRNA and the non-targeting contrast siRNA Qneg each, with 5nM, 20nM and 100nM concentration, with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) mix, and be used for the Vero cell of transfection inoculation.After the transfection, with 0.1MOI (infection multiplicity) PR8 influenza infection cell.For quantitative, by hemagglutinin (HA) analytic process the supernatant that obtains from the hole that contains infection cell is tested, as described in embodiment 7 virus that produces.Table 7 has been summed up the strike low activity of every kind of siRNA convection current sensillary base because of expressing, and represents (1-(HA units/usefulness of handling with influenza siRNA contrasts the HA units that siRNA handles) with the minimizing percentage rate of virus titer.

Table 7

With influenza infection transfection siRNA

The Vero cell in the minimizing percentage rate of virus titer

SiRNA concentration	DX2852	DX2855	DX2858	DX2861	DX2956	G1498
							100nM
	0	25	0	15	50	50
							20nM	25	15	40	0	50	50
5nM	40	25	0	40	50	50

Data show influenza specificity dicer substrate siRNA DX2956 in the table 7 has reduced effectively with the influenza titre in the Vero cell of influenza infection.DX2956 has shown the activity similar as G1498, and other four kinds of dicer substrate siRNA demonstrate the activity still less than DX2956.These digital proofs, when with Lipofectamine 2000 (Invitrogen, Carlsbad, Ca) during transfection, the outstanding candidate that siDNA DX2956 effectively reduces influenza mRNA level and therefore infects for treatment or flu-prevention.

Though it is clearer in order to understand, aforesaid invention at length is described in the mode of embodiment, but the technical staff be it is apparent that some variation and modifies in the scope of the claim of enclosing that can present in the mode with example rather than restriction and implement.In context, various publications and other list of references are cited in foregoing disclose to save the mode of describing.Each piece of writing of these lists of references is all purposes, by reference this by complete being included in.Yet, it should be noted that involved at the various publications of this discussion only is before applying date in present patent application because of disclosing of they, and the inventor keeps the right of this open request priority according to previous invention.

Sequence table

＜110〉Nastech Pharm Co.

＜120〉as the peptide-DICER substrate RNA conjugates of SIRNA delivery vehicle

<130>05-20PCT

<140>

<141>

<150>60/733,665

<151>2005-04-08

<150>60/822,896

<151>2006-08-18

<160>166

<170>PatentIn Ver.3.3

<210>1

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>1

Lys Arg Arg Gln Arg Arg Arg

1 5

<210>2

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>2

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>3

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>3

Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr

1 5 10 15

Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro

20 25 30

Val Asp

<210>4

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>4

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

<210>5

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>5

Ala Ala Val Leu Leu Pro Val Leu Leu Pro Val Leu Leu Ala Ala Pro

1 5 10 15

<210>6

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>6

Val Thr Val Leu Ala Leu Gly Ala Leu Ala Gly Val Gly Val Gly

1 5 10 15

<210>7

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>7

Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly

1 5 10 15

Ala

<210>8

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>8

Met Gly Leu Gly Leu His Leu Leu Val Leu Ala Ala Ala Leu Gln Gly

1 5 10 15

Ala

<210>9

<211>24

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>9

Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln

1 5 10 15

Thr Ala Ile Gly Val Gly Ala Pro

20

<210>10

<211>26

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>10

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys

1 5 10 15

Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

20 25

<210>11

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>11

Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Lys Lys Lys Lys

1 5 10 15

<210>12

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>12

Arg Arg Arg Arg Arg Arg Arg

1 5

<210>13

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>13

Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys

1 5 10 15

Leu Ala

<210>14

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>14

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

<210>15

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>15

Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly Val Ala Thr Ala

1 5 10 15

<210>16

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>16

Phe Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val

1 5 10 15

<210>17

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>17

Gly Val Phe Val Leu Gly Phe Leu Gly Phe Leu Ala Thr Ala Gly Ser

1 5 10 15

<210>18

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>18

Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala

1 5 10 15

<210>19

<211>56

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>19

Ala Cys Thr Cys Pro Tyr Cys Lys Asp Ser Glu Gly Arg Gly Ser Gly

1 5 10 15

Asp Pro Gly Lys Lys Lys Gln His Ile Cys His Ile Gln Gly Cys Gly

20 25 30

Lys Val Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Trp His

35 40 45

Thr Gly Glu Arg Pro Phe Met Cys

50 55

<210>20

<211>54

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>20

Ala Cys Thr Cys Pro Asn Cys Lys Asp Gly Glu Lys Arg Ser Gly Glu

1 5 10 15

Gln Gly Lys Lys Lys His Val Cys His Ile Pro Asp Cys Gly Lys Thr

20 25 30

Phe Arg Lys Thr Ser Leu Leu Arg Ala His Val Arg Leu His Thr Gly

35 40 45

Glu Arg Pro Phe Val Cys

50

<210>21

<211>55

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>21

Ala Cys Thr Cys Pro Asn Cys Lys Glu Gly Gly Gly Arg Gly Thr Asn

1 5 10 15

Leu Gly Lys Lys Lys Gln His Ile Cys His Ile Pro Gly Cys Gly Lys

20 25 30

Val Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Trp His Ser

35 40 45

Gly Glu Arg Pro Phe Val Cys

50 55

<210>22

<211>56

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>22

Ala Cys Ser Cys Pro Asn Cys Arg Glu Gly Glu Gly Arg Gly Ser Asn

1 5 10 15

Glu Pro Gly Lys Lys Lys Gln His Ile Cys His Ile Glu Gly Cys Gly

20 25 30

Lys Val Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Trp His

35 40 45

Thr Gly Glu Arg Pro Phe Ile Cys

50 55

<210>23

<211>60

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>23

Arg Cys Thr Cys Pro Asn Cys Thr Asn Glu Met Ser Gly Leu Pro Pro

1 5 10 15

Ile Val Gly Pro Asp Glu Arg Gly Arg Lys Gln His Ile Cys His Ile

20 25 30

Pro Gly Cys Glu Arg Leu Tyr Gly Lys Ala Ser His Leu Lys Thr His

35 40 45

Leu Arg Trp His Thr Gly Glu Arg Pro Phe Leu Cys

50 55 60

<210>24

<211>58

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>24

Thr Cys Asp Cys Pro Asn Cys Gln Glu Ala Glu Arg Leu Gly Pro Ala

1 5 10 15

Gly Val His Leu Arg Lys Lys Asn Ile His Ser Cys His Ile Pro Gly

20 25 30

Cys Gly Lys Val Tyr Gly Lys Thr Ser His Leu Lys Ala His Leu Arg

35 40 45

Trp His Thr Gly Glu Arg Pro Phe Val Cys

50 55

<210>25

<211>53

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>25

Arg Cys Thr Cys Pro Asn Cys Lys Ala Ile Lys His Gly Asp Arg Gly

1 5 10 15

Ser Gln His Thr His Leu Cys Ser Val Pro Gly Cys Gly Lys Thr Tyr

20 25 30

Lys Lys Thr Ser His Leu Arg Ala His Leu Arg Lys His Thr Gly Asp

35 40 45

Arg Pro Phe Val Cys

50

<210>26

<211>56

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>26

Pro Gln Ile Ser Leu Lys Lys Lys Ile Phe Phe Phe Ile Phe Ser Asn

1 5 10 15

Phe Arg Gly Asp Gly Lys Ser Arg Ile His Ile Cys His Leu Cys Ash

20 25 30

Lys Thr Tyr Gly Lys Thr Ser His Leu Arg Ala His Leu Arg Gly His

35 40 45

Ala Gly Asn Lys Pro Phe Ala Cys

50 55

<210>27

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>27

Trp Trp Glu Thr Trp Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn

1 5 10 15

Phe Ser Thr Arg Gln Ala Arg Arg Asn His Arg Arg Arg His Arg

20 25 30

<210>28

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>28

Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

1 5 10 15

<210>29

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>29

Arg Val Ile Arg Val Trp Phe Gln Asn Lys Arg Cys Lys Asp Lys Lys

1 5 10 15

<210>30

<211>39

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>30

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Arg Lys

1 5 10 15

Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Arg Lys Lys Arg Arg

20 25 30

Gln Arg Arg Arg Pro Pro Gln

35

<210>31

<211>22

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>31

Gly Glu Gln Ile Ala Gln Leu Ile Ala Gly Tyr Ile Asp Ile Ile Leu

1 5 10 15

Lys Lys Lys Lys Ser Lys

20

<210>32

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(2)..(5)

＜223〉variable amino acid

<220>

<221>MOD_RES

<222>(7)..(18)

＜223〉variable amino acid

<220>

<221>MOD_RES

<222>(20)..(22)

＜223〉variable amino acid

<400>32

Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa His Xaa Xaa Xaa His

20

<210>33

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>33

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>34

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>34

ataagatcct t 11

<210>35

<211>27

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>35

augguguggg ugaggagcac augggug 27

<210>36

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>36

cccaugugcu ccucacccac accat 25

<210>37

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>37

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>38

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>38

ggatcttatt tcttcggagt t 21

<210>39

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>39

ctccgaagaa 10

<210>40

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>40

ataagatcct t 11

<210>41

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>41

Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5 10 15

<210>42

<211>33

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>42

Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys

1 5 10 15

Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys

20 25 30

Gln

<210>43

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>43

Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg

1 5 10 15

Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

20 25 30

<210>44

<211>27

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>44

Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser

1 5 10 15

Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

20 25

<210>45

<211>24

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>45

Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val

1 5 10 15

Tyr Val Tyr Lys Val Leu Lys Gln

20

<210>46

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>46

Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr

1 5 10 15

Lys Val Leu Lys Gln

20

<210>47

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>47

Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu

1 5 10 15

Lys Gln

<210>48

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>48

Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5 10

<210>49

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>49

Val Tyr Val Tyr Lys Val Leu Lys Gln

1 5

<210>50

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>50

Tyr Lys Val Leu Lys Gln

1 5

<210>51

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>51

Lys Val Leu Lys Gln

1 5

<210>52

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>52

Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Glu Gly Lys

1 5 10 15

Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys

20 25 30

Val Leu Lys Gln

35

<210>53

<211>21

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>53

gccuguaccu caucuacucu u 21

<210>54

<211>21

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>54

gaguagauga gguacaggcu u 21

<210>55

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>55

gccucuucuc cuuccugauc guggc 25

<210>56

<211>27

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>56

gccacgauca ggaaggagaa gaggcug 27

<210>57

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>57

gccugcugca cuuuggagug aucgg 25

<210>58

<211>27

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>58

ccgaucacuc caaagugcag caggcag 27

<210>59

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>59

cccaugugcu ccucacccac accat 25

<210>60

<211>27

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>60

augguguggg ugaggagcac augggug 27

<210>61

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>61

accucaucua cucccagguc cuctt 25

<210>62

<211>27

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>62

aagaggaccu gggaguagau gagguac 27

<210>63

<211>27

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>63

gccuguaccu caucuacucc caggucc 27

<210>64

<211>29

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>64

gguccuggga guagaugagg uacaggcuu 29

<210>65

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>65

agacagcgac caaaagaauu cggau 25

<210>66

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>66

auccgaauuc uuuuggucgc ugucutt 27

<210>67

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>67

augaagaucu guuccaccau ugaag 25

<210>68

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>68

cuucaauggu ggaacagauc uucautt 27

<210>69

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>69

gaucuguucc accauugaag aacuc 25

<210>70

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>70

gaguucuuca augguggaac agauctt 27

<210>71

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>71

uugaggagug ccugauuaau gaucc 25

<210>72

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>72

ggaucauuaa ucaggcacuc cucaatt 27

<210>73

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>73

ggaucuuauu ucuucggaga caatg 25

<210>74

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>74

cauugucucc gaagaaauaa gaucctt 27

<210>75

<211>25

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>75

ggaucuuauu ucuucggaga caaug 25

<210>76

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>76

cauugucucc gaagaaauaa gaucctt 27

<210>77

<211>6

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>77

ggaucu 6

<210>78

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>78

auuucuucgg agacaaug 18

<210>79

<211>8

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>79

ggaucuua 8

<210>80

<211>16

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>80

uucuucggag acaaug 16

<210>81

<211>10

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>81

ggaucuuauu 10

<210>82

<211>14

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>82

cuucggagac aaug 14

<210>83

<211>12

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>83

ggaucuuauu uc 12

<210>84

<211>12

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>84

ucggagacaa ug 12

<210>85

<211>14

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>85

ggaucuuauu ucuu 14

<210>86

<211>10

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>86

ggagacaaug 10

<210>87

<211>16

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>87

ggaucuuauu ucuucg 16

<210>88

<211>8

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>88

agacaaug 8

<210>89

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>89

ggaucuuauu ucuucgga 18

<210>90

<211>6

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>90

acaaug 6

<210>91

<211>6

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>91

ggaucu 6

<210>92

<211>17

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>92

uuucuucgga gacaaug 17

<210>93

<211>8

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>93

ggaucuua 8

<210>94

<211>15

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>94

ucuucggaga caaug 15

<210>95

<211>10

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>95

ggaucuuauu 10

<210>96

<211>13

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>96

uucggagaca aug 13

<210>97

<211>12

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>97

ggaucuuauu uc 12

<210>98

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>98

cggagacaau g 11

<210>99

<211>14

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>99

ggaucuuauu ucuu 14

<210>100

<211>9

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>100

gagacaaug 9

<210>101

<211>16

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>101

ggaucuuauu ucuucg 16

<210>102

<211>7

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>102

gacaaug 7

<210>103

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>103

ggaucuuauu ucuucgga 18

<210>104

<211>5

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>104

caaug 5

<210>105

<211>6

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>105

ggaucu 6

<210>106

<211>15

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>106

ucuucggaga caaug 15

<210>107

<211>8

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>107

ggaucuua 8

<210>108

<211>13

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>108

uucggagaca aug 13

<210>109

<211>10

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>109

ggaucuuauu 10

<210>110

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>110

cggagacaau g 11

<210>111

<211>12

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>111

ggaucuuauu uc 12

<210>112

<211>9

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>112

gagacaaug 9

<210>113

<211>14

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>113

ggaucuuauu ucuu 14

<210>114

<211>7

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>114

gacaaug 7

<210>115

<211>16

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>115

ggaucuuauu ucuucg 16

<210>116

<211>5

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>116

caaug 5

<210>117

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>117

ggaucuuauu ucuucgga 18

<210>118

<211>3

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>118

aug 3

<210>119

<211>6

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>119

ggaucu 6

<210>120

<211>13

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>120

uucggagaca aug 13

<210>121

<211>8

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>121

ggaucuua 8

<210>122

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>122

cggagacaau g 11

<210>123

<211>10

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>123

ggaucuuauu 10

<210>124

<211>9

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>124

gagacaaug 9

<210>125

<211>12

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>125

ggaucuuauu uc 12

<210>126

<211>7

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>126

gacaaug 7

<210>127

<211>14

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>127

ggaucuuauu ucuu 14

<210>128

<211>5

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>128

caaug 5

<210>129

<211>16

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>129

ggaucuuauu ucuucg 16

<210>130

<211>3

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>130

aug 3

<210>131

<211>18

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>131

ggaucuuauu ucuucgga 18

<210>132

<211>1

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>132

g 1

<210>133

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>133

ggaucuuauu ucuucggagt t 21

<210>134

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>134

cuccgaagaa auaagaucct t 21

<210>135

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>135

ggaucuuauu u 11

<210>136

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>136

cuucggagtt 10

<210>137

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>137

cuccgaagaa auaagaucct t 21

<210>138

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>138

ggaucuuauu u 11

<210>139

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>139

cuucggagtt 10

<210>140

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>140

cuccgaagaa auaagaucct t 21

<210>141

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>141

ggatcttatt t 11

<210>142

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>142

cttcggagtt 10

<210>143

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>143

ctccgaagaa ataagatcct t 21

<210>144

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>144

ggatcttatt t 11

<210>145

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>145

cttcggagtt 10

<210>146

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>146

ctccgaagaa ataagatcct t 21

<210>147

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>147

ggatcttatt tcttcggagt t 21

<210>148

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>148

ctccgaagaa 10

<210>149

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>149

ataagatcct t 11

<210>150

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>150

ggatcttatt tcttcggagt t 21

<210>151

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>151

ctccgaagaa 10

<210>152

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>152

ataagatcct t 11

<210>153

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>153

ggaucuuauu u 11

<210>154

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>154

cuucggagtt 10

<210>155

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>155

cuccgaagaa auaagaucct t 21

<210>156

<211>11

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>156

ggaucuuauu u 11

<210>157

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>157

cuucggagtt 10

<210>158

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of bonded DNA/RNA molecule: synthetic oligonucleotide

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>158

cuccgaagaa auaagaucct t 21

<210>159

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>159

ggatcttatt t 11

<210>160

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>160

cttcggagtt 10

<210>161

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>161

ctccgaagaa ataagatcct t 21

<210>162

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>162

ggatcttatt t 11

<210>163

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>163

cttcggagtt 10

<210>164

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>164

ctccgaagaa ataagatcct t 21

<210>165

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>165

ggatcttatt tcttcggagt t 21

<210>166

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>166

ctccgaagaa 10

Claims (according to the modification of the 19th of treaty)

1. compositions, it comprises:

(a) double stranded RNA (dsRNA) molecule, wherein said chain have about 25 to the length of about 30 nucleotide, and described nucleotide can be identical or different; With

(b) comprise about 5 to about 40 amino acid whose peptides, wherein said peptide contains aminoacid sequence KVLKQ (SEQ ID NO:51);

Wherein said dsRNA molecule conjugates to described peptide.

2. compositions according to claim 1, wherein said dsRNA molecule is siRNA.

3. compositions according to claim 2, wherein said siRNA contains with the nucleotide sequence that comes from the part of human TNF-α gene nucleic acid sequence.

4. compositions according to claim 2, wherein said siRNA contains with the nucleotide sequence that comes from the part of viral genomic nucleic acid sequence.

5. compositions according to claim 4, the source of wherein said viral gene are influenza virus.

6. compositions according to claim 1, it further comprises carrier.

7. compositions according to claim 1, wherein said dsRNA molecule comprise that further the strand 3 ' antisense strand that contains 2 nucleotide dangles.

8. compositions according to claim 1, wherein said dsRNA molecule comprise that further the strand 3 ' sense strand that contains 2 nucleotide dangles.

9. compositions according to claim 1, wherein said dsRNA molecule not tool dangles.

10. compositions according to claim 1, wherein said chain have about 25 to the length of about 29 nucleotide, and described nucleotide can be identical or different.

11. compositions according to claim 1, wherein said dsRNA molecule are by being made up of adopted RNA chain and antisense RNA chain, and described peptide conjugates to 5 of described antisense RNA chain ' end.

12. compositions according to claim 1, the described aminoacid sequence of wherein said peptide is selected from down group:

KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：33)；

KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：42)；

VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：43)；

AQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：44)；

KDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：45)；

KKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：46)；

KRSRKESYSVYVYKVLKQ(SEQ ID NO：47)；

RKESYSVYVYKVLKQ(SEQ ID NO：41)；

SYSVYVYKVLKQ(SEQ ID NO：48)；VYVYKVLKQ(SEQ ID NO：49)；

YKVLKQ (SEQ ID NO:50); And KVLKQ (SEQ ID NO:51).

13. compositions according to claim 1, wherein said peptide conjugates to the molecule in conjunction with zooblast.

14. be used to improve the application of the inflammation relevant with TNF-α according to the described compositions of arbitrary claim among claim 1-3 or the 6-13, it comprises that the described compositions with the improvement amount delivers medicine to animal.

15. be used for improving application in the medicament of the animal inflammation relevant in preparation with TNF-α according to the described compositions of arbitrary claim among claim 1-3 or the 6-13.

16. according to claim 14 or 15 described application, wherein said inflammation occurs in the arthritis.

17. according to claim 14 or 15 described application, wherein said inflammation occurs in the psoriasis.

18. pharmaceutical composition be used for suppressor gene in the expression of animal to improve the application of inflammation, it comprises double stranded RNA (dsRNA) molecule is administered into described animal, wherein said pharmaceutical composition comprises described dsRNA molecule and peptide, wherein said dsRNA molecule comprises about 25 to about 30 base pairs, wherein said peptide comprises about 5 to about 40 aminoacid and comprise aminoacid sequence KVLKQ (SEQ ID NO:51), and wherein said dsRNA molecule conjugates to described peptide.

19. comprising the pharmaceutical composition of double stranded RNA (dsRNA) molecule and peptide expresses in animal with the application in the medicament that improves inflammation at the preparation suppressor gene, wherein said dsRNA molecule comprises about 25 to about 30 base pairs, wherein said peptide comprises about 5 to about 40 aminoacid and comprise aminoacid sequence KVLKQ (SEQ ID NO:51), and wherein said dsRNA molecule conjugates to described peptide.

20. according to claim 18 or 19 described application, wherein said inflammation occurs in the arthritis.

21. according to claim 18 or 19 described application, wherein said inflammation occurs in the psoriasis.

22. compositions according to claim 1 is used to improve the application of the infection relevant with influenza virus, it comprises that the described compositions with the improvement amount is administered into animal.

23. compositions according to claim 1 is used for improving application in the medicament of the animal infection relevant with influenza virus in preparation.

24. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule,

Described siRNA molecule comprise about 15 nucleotide to article one RNA chain (A chain) of about 50 nucleotide, about 1 nucleotide to about 25 nucleotide second RNA chain (B1 chain) and about 1 nucleotide to the 3rd the RNA chain (B2 chain) of about 25 nucleotide;

Wherein said dsRNA molecule conjugates to peptide;

The complementary Non-overlapping Domain of each bar in wherein said B1 chain and the described B2 chain to described A chain;

Wherein first double-stranded region (A:B1) is by described B1 chain of annealing and described A chain formation; With

Wherein second double-stranded region (A:B2) is by described B2 chain of annealing and described A chain formation; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

25. compositions according to claim 24, wherein said A:B1 duplex separates from described A:B2 duplex by otch or breach, wherein said breach produces at least one unpaired nucleotide in described A chain, and described unpaired nucleotide is between described A:B1 duplex and described A:B2 duplex.

26. compositions according to claim 25, it further comprises one or more unpaired nucleotide, the 3 ' end of described unpaired nucleotide on described A chain, described B1 chain and/or described B2 chain one or two.

27. according to the described compositions of arbitrary claim among the claim 25-26, wherein said A chain is that about 18 nucleotide are to about 40 nucleotide.

28. compositions according to claim 27, wherein said A chain are that about 20 nucleotide are to about 32 nucleotide.

29. compositions according to claim 28, wherein said A chain are 21,22,23,24,25,26,27,28,29,30 or 31 nucleotide.

30. according to the described compositions of arbitrary claim among the claim 25-26, wherein said A:B1 duplex and described A:B2 duplex comprise that altogether about 15 base pairs are to about 40 base pairs.

31. compositions according to claim 30, wherein said A:B1 duplex and described A:B2 duplex comprise that altogether about 18 base pairs are to about 35 base pairs.

32. compositions according to claim 31, wherein said A:B1 duplex and described A:B2 duplex comprise that altogether about 20 base pairs are to about 30 base pairs.

33. compositions according to claim 32, wherein said A:B1 duplex and described A:B2 duplex comprise 21,22,23,24,25,26,27,28 or 29 base pairs altogether.

34. compositions according to claim 26, wherein said siRNA molecule comprise the strand 3 of one or two 1 nucleotide to 5 nucleotide ' dangle.

35. according to the described compositions of arbitrary claim among the claim 25-26, wherein said A chain comprises nucleotide sequence 3 ' TTCCUAGAAUAAAGAAGCCUCUGUUAC5 ' (SEQID NO:76).

36. compositions according to claim 22, wherein said B1 chain comprise nucleotide sequence 5 ' GGAUCU3 ' (SEQ ID NO:77).

37. compositions according to claim 25, wherein said B2 chain comprise nucleotide sequence 5 ' ACAAUG3 ' (SEQ ID NO:90).

38. compositions according to claim 25, wherein said A:B1 duplex separates from described A:B2 duplex by otch.

39. according to the described compositions of claim 38, wherein said B2 chain stops with 5 ' hydroxyl.

40. according to the described compositions of claim 38, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:135), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:136), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:137).

41. according to the described compositions of claim 38, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:144), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:145), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:146).

42. according to the described compositions of claim 38, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:148), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:149), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATT TCTTCGGAGTT3 ' (SEQ ID NO:147).

43. according to the described compositions of claim 38, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:153), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:154), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:155).

44. according to the described compositions of claim 38, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:159), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:160), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:161).

45. according to the described compositions of claim 38, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:166), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:34), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATT TCTTCGGAGTT3 ' (SEQ ID NO:165).

46. according to the described compositions of claim 38, wherein said B1 chain stops with 5 ' phosphoric acid.

47. according to the described compositions of claim 46, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:138), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:139), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:140).

48. according to the described compositions of claim 46, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:141), wherein said B2 chain comprises nucleotide sequence 5 ' TTCGGAGTT3 ' (SEQ ID NO:142), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:143).

49. according to the described compositions of claim 46, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:151), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:152), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATTTCTTCGGAGTT3 ' (SEQ ID NO:150).

50. according to the described compositions of claim 46, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:156), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:157), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:158).

51. according to the described compositions of claim 46, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:162), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:163), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAAATAAGATCCTT3 ' (SEQ ID NO:164).

52. according to the described compositions of claim 46, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:39), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:40), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATTTCTTCGGAGTT3 ' (SEQ ID NO:38).

53. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1B2);

Wherein A:B1B2 comprises that about altogether 14 base pairs arrive about 24 base pairs altogether;

Wherein A represents sense strand and B1B2 represents antisense strand;

Wherein A is that about 19 nucleotide are to about 25 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide; With

Wherein the pattern length of B1 and B2 is that about 13 nucleotide are to about 23 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

54. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1B2);

Wherein A:B1B2 comprises that about altogether 16 base pairs arrive about 22 base pairs altogether;

Wherein A represents sense strand and B1B2 represents antisense strand;

Wherein A is that about 19 nucleotide are to about 23 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide; With

Wherein the pattern length of B1 and B2 is that about 13 nucleotide are to about 23 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

55. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1B2);

Wherein A:B1B2 comprises that about altogether 14 base pairs arrive about 24 base pairs altogether;

Wherein A represents antisense strand and B1B2 represents sense strand;

Wherein A is that about 14 nucleotide are to about 24 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide; With

Wherein the pattern length of B1 and B2 is that about 18 nucleotide are to about 24 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

56. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1B2);

Wherein A:B1B2 comprises that about altogether 14 base pairs arrive about 22 base pairs altogether;

Wherein A represents antisense strand and B1B2 represents sense strand;

Wherein A is that about 16 nucleotide are to about 22 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide;

Wherein the pattern length of B1 and B2 is that about 18 nucleotide are to about 22 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

57. a compositions, it comprises:

(a) according to the described siRNA molecule of arbitrary claim among claim 24 and the 53-56, wherein said siRNA molecule reduces the titre of the target virus that is selected from the group of being made up of retrovirus, respiratory syncytial virus, human metapneumovirus, human parainfluenza virus and influenza virus effectively; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

58. according to the described compositions of arbitrary claim among claim 24 and the 53-56, wherein said peptide comprises the aminoacid sequence that is selected from by the following group of forming: KRRQRRR (SEQ ID NO:1), RQIKIWFQNRRMKWKK (SEQ ID NO:2), DAATATRGRSAASRPTERPRAPARSASRPRRPVD (SEQ ID NO:3), AAVALLPAVLLALLAP (SEQ ID NO:4), AAVLLPVLLPVLLAAP (SEQID NO:5), VTVLALGALAGVGVG (SEQ ID NO:6), GALFLGWLGAAGSTMGA (SEQ ID NO:7), MGLGLHLLVLAAALQGA (SEQ ID NO:8), LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO:9), GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO:10), TPPKKKRKVEDPKKKK (SEQ ID NO:11), RRRRRRR (SEQ ID NO:12), KLALKLALKALKAALKLA (SEQ ID NO:13), GLFGAIAGFIENGWEG (SEQ ID NO:14), FFGAVIGTIALGVTA (SEQID NO:15), FLGFLLGVGSAIASGV (SEQ ID NO:16), GVFVLGFLGFLATAGS (SEQ ID NO:17), GAALGLAWIPYFGPAA (SEQID NO:18), ACTCPYCKDSEGRGSGDPGKKKQHICHIQGCGKVYGKTSHLRAHLRWHTGERPFMC (SEQ ID NO: 19), ACTCPNCKDGEKRSGEQGKKKHVCHIPDCGKTFRKTSLLRAHVRLHTGERPFVC (SEQ ID NO:20), ACTCPNCKEGGGRGTNLGKKKQHICHIPGCGKVYGKTSHLRAHLRWHSGERPFVC (SEQ ID NO:21), ACSCPNCREGEGRGSNEPGKKKQHICHIEGCGKVYGKTSHLRAHLRWHTGERPFIC (SEQ ID NO:22), RCTCPNCTNEMSGLPPIVGPDERGRKQHICHIPGCERLYGKASHLKTHLRWHTGER PFLC (SEQ ID NO:23), TCDCPNCQEAERLGPAGVHLRKKNIHSCHIPGCGKVYGKTSHLKAHLRWHTGERPF VC (SEQ ID NO:24), RCTCPNCKAIKHGDRGSQHTHLCSVPGCGKTYKKTSHLRAHLRKHTGDRPFVC (SEQ ID NO:25), PQISLKKKIFFFIFSNFRGDGKSRIHICHLCNKTYGKT SHLRAHLRGHAGNKPFAC (SEQ ID NO:26), WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO:27), GKINLKALAALAKKIL (SEQ ID NO:28), RVIRVWFQNKRCKDKK (SEQID NO:29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO:30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO:31), KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:33), KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ IDNO:37), RKESYSVYVYKVLKQ (SEQ ID NO:41), KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:42), VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:43), AQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:44), KDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:45), KKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:46), KRSRKESYSVYVYKVLKQ (SEQ ID NO:47), SYSVYVYKVLKQ (SEQID NO:48), VYVYKVLKQ (SEQ ID NO:49), YKVLKQ (SEQ ID NO:50), KVLKQ (SEQ ID NO:51) and KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:52).

59.siRNA molecule is used to reduce the application of target virus titer, described application may further comprise the steps:

(a) target gene of the gene silencing of selection siRNA mediation, wherein said target gene is the target viral gene;

(b) design and/or synthetic suitable siRNA molecule are used for the gene silencing of the siRNA mediation of described target viral gene, and each in the wherein said siRNA molecule includes the duplex of breach or otch and wherein said breach or otch and appears in the sense strand or antisense strand of described siRNA duplex;

(c) yoke closes described siRNA to comprising about 5 to about 40 amino acid whose peptides; With

(d) described siRNA molecule is administered to the cell of expressing described target viral gene,

Wherein this siRNA molecular peptide conjugates can specificity in conjunction with corresponding target virus mRNA thereby reduce its expression in cell.

60.siRNA molecule is used to prepare the application that reduces the medicament of target virus titer by the gene silencing of the siRNA mediation of target viral gene, each in the wherein said siRNA molecule includes the duplex of breach or otch and wherein said breach or otch and appears in the sense strand or antisense strand of described siRNA duplex; Wherein said siRNA conjugates to and comprises about 5 to about 40 amino acid whose peptides; And wherein this siRNA molecular peptide conjugates can specificity in conjunction with corresponding target virus mRNA thereby reduce its expression in cell.

61.siRNA molecule is used to reduce the application that endogenous gene is expressed, described application may further comprise the steps:

(a) target gene of the gene silencing of selection siRNA mediation, wherein said target gene is an endogenous gene;

(b) the duplex siRNA molecule of design and/or synthetic suitable jagged or otch is used for the gene silencing of the siRNA mediation of described endogenous target gene, and wherein said siRNA molecule includes the duplex of breach or otch and wherein said breach or otch and appears in the sense strand or antisense strand of described siRNA molecule; With

(c) yoke closes described siRNA to comprising about 5 to about 40 amino acid whose peptides; With

(d) described siRNA molecule is administered to the cell of expressing described endogenous target gene,

Wherein this siRNA molecular peptide conjugates can specificity in conjunction with corresponding endogenous said target mrna thereby reduce its expression in cell.

62.siRNA molecule is used to prepare the application that gene silencing by the siRNA mediation of endogenous target gene reduces the medicament that endogenous gene expresses, wherein said siRNA molecule includes the duplex of breach or otch and wherein said breach or otch and appears in the sense strand or antisense strand of described siRNA molecule; And wherein said siRNA conjugates to and comprises about 5 to about 40 amino acid whose peptides; And wherein this siRNA molecular peptide conjugates can specificity in conjunction with corresponding endogenous said target mrna thereby reduce its expression in cell.

63. according to the described compositions of arbitrary claim among claim 1-13 or the 24-58, it is as medicine.

64. according to the described compositions of arbitrary claim among claim 1-3 or the 6-13, it is as the medicament of improvement with TNF-α related inflammation.

65. according to the described compositions of claim 64, wherein said inflammation occurs in the arthritis.

66. according to the described compositions of claim 64, wherein said inflammation occurs in the psoriasis.

67. compositions according to claim 1, it is as the medicament of improvement with the influenza virus infections relating.

Claims (65)

1. compositions, it comprises:

(a) double stranded RNA (dsRNA) molecule, wherein said chain have about 25 to the length of about 30 base pairs, and described base pair can be identical or different; With

(b) comprise about 5 to about 40 amino acid whose peptides, wherein said peptide contains aminoacid sequence KVLKQ (SEQ ID NO:51);

Wherein said dsRNA molecule conjugates to described peptide.

2. compositions according to claim 1, wherein said dsRNA is siRNA.

3. compositions according to claim 2, wherein said siRNA contains with the sequence that comes from the part of human TNF-α gene order.

4. compositions according to claim 2, wherein said siRNA contains with the sequence that comes from the part of virus gene sequence.

5. compositions according to claim 4, the source of wherein said viral gene are influenza virus.

6. compositions according to claim 1, it further comprises carrier.

7. compositions according to claim 1, wherein said dsRNA has 2bp 3 ' antisense strand and dangles.

8. compositions according to claim 1, wherein said dsRNA has 2bp 3 sense strands and dangles.

9. compositions according to claim 1, wherein said dsRNA not tool dangles.

10. compositions according to claim 1, wherein said chain have about 25 to the length of about 29 base pairs, and described base pair can be identical or different.

11. compositions according to claim 1, wherein said dsRNA molecule are by being made up of adopted RNA chain and antisense RNA chain, and described peptide conjugates to 5 of described antisense strand ' end.

12. compositions according to claim 1, the described aminoacid sequence of wherein said peptide is selected from down group:

KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：33)；

KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：42)；

VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：43)；

AQKKDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：44)；

KDGKKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：45)；

KKRKRSRKESYSVYVYKVLKQ(SEQ ID NO：46)；

KRSRKESYSVYVYKVLKQ(SEQ ID NO：47)；

RKESYSVYVYKVLKQ(SEQ ID NO：41)；

SYSVYVYKVLKQ(SEQ ID NO：48)；VYVYKVLKQ(SEQ ID NO：49)；

YKVLKQ (SEQ ID NO:50); And KVLKQ (SEQ ID NO:51).

13. compositions according to claim 1, wherein said peptide conjugates to the molecule in conjunction with zooblast.

14. a method that is used to improve the inflammation relevant with TNF-α, it comprises that each the described compositions of claim 1-13 with the improvement amount delivers medicine to animal.

15. method according to claim 14, wherein said inflammation occurs in the arthritis.

16. method according to claim 14, wherein said inflammation occurs in the psoriasis.

17. one kind is used for suppressor gene and expresses to improve the method for inflammation animal, it comprises that the compositions with double stranded RNA (dsRNA) molecule is administered into described animal, wherein this pharmaceutical compositions comprises described dsRNA molecule and peptide, wherein said dsRNA molecule comprises about 25 to about 30 base pairs, and wherein said peptide comprises about 5 to about 40 aminoacid and comprise aminoacid sequence KVLKQ (SEQ ID NO:51); And wherein said dsRNA molecule conjugates to described peptide.

18. method according to claim 17, wherein said inflammation occurs in the arthritis.

19. method according to claim 17, wherein said inflammation occurs in the psoriasis.

20. a method that is used to improve with the influenza virus infections relating, it comprises that the compositions according to claim 1 with the improvement amount is administered into animal.

21. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule,

Described siRNA molecule comprise about 15 nucleotide to article one RNA chain (A chain) of about 50 nucleotide, about 1 nucleotide to about 25 nucleotide second RNA chain (B1 chain) and about 1 nucleotide to the 3rd the RNA chain (B2 chain) of about 25 nucleotide;

Wherein said dsRNA molecule conjugates to peptide;

The complementary Non-overlapping Domain of each bar in wherein said B1 chain and the described B2 chain to described A chain;

Wherein first double-stranded region (A:B1) is by described B1 chain of annealing and described A chain formation; With

Wherein second double-stranded region (A:B2) is by described B2 chain of annealing and described A chain formation; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

22. compositions according to claim 21, wherein said A:B1 duplex separates from described A:B2 duplex by otch or breach, wherein said breach produces at least one unpaired nucleotide in described A chain, and described unpaired nucleotide is between described A:B1 duplex and described A:B2 duplex.

23. compositions according to claim 22, it further comprises one or more unpaired nucleotide, the 3 ' end of described unpaired nucleotide on described A chain, described B1 chain and/or described B2 chain one or two.

24. according to the described compositions of arbitrary claim among the claim 22-23, wherein said A chain is that about 18 nucleotide are to about 40 nucleotide.

25. compositions according to claim 24, wherein said A chain are that about 20 nucleotide are to about 32 nucleotide.

26. compositions according to claim 25, wherein said A chain are 21,22,23,24,25,26,27,28,29,30 or 31 nucleotide.

27. according to the described compositions of arbitrary claim among the claim 22-23, wherein said A:B1 duplex and described A:B2 duplex comprise that altogether about 15 base pairs are to about 40 base pairs.

28. compositions according to claim 27, wherein said A:B1 duplex and described A:B2 duplex comprise that altogether about 18 base pairs are to about 35 base pairs.

29. compositions according to claim 28, wherein said A:B1 duplex and described A:B2 duplex comprise that altogether about 20 base pairs are to about 30 base pairs.

30. compositions according to claim 29, wherein said A:B1 duplex and described A:B2 duplex comprise 21,22,23,24,25,26,27,28 or 29 base pairs altogether.

31. compositions according to claim 23, wherein said siRNA molecule comprise the strand 3 of one or two 1 nucleotide to 5 nucleotide ' dangle.

32. according to the described compositions of arbitrary claim among the claim 22-23, wherein said A chain comprises nucleotide sequence 3 ' TTCCUAGAAUAAAGAAGCCUCUGUUAC5 ' (SEQID NO:76).

33. compositions according to claim 22, wherein said B1 chain comprise nucleotide sequence 5 ' GGAUCU3 ' (SEQ ID NO:77).

34. compositions according to claim 22, wherein said B2 chain comprise nucleotide sequence 5 ' ACAAUG3 ' (SEQ ID NO:90).

35. compositions according to claim 22, wherein said A:B1 duplex separates from described A:B2 duplex by otch.

36. compositions according to claim 35, wherein said B2 chain stops with 5 ' hydroxyl.

37. compositions according to claim 35, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:135), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:136), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:137).

38. compositions according to claim 35, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:144), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:145), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:146).

39. compositions according to claim 35, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:148), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:149), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATT TCTTCGGAGTT3 ' (SEQ ID NO:147).

40. compositions according to claim 35, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:153), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:154), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:155).

41. compositions according to claim 35, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:159), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:160), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:161).

42. compositions according to claim 35, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:166), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:34), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATT TCTTCGGAGTT3 ' (SEQ ID NO:165).

43. compositions according to claim 35, wherein said B1 chain stops with 5 ' phosphoric acid.

44. according to the described compositions of claim 43, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:138), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:139), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:140).

45. according to the described compositions of claim 43, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:141), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:142), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:143).

46. according to the described compositions of claim 43, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:151), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:152), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATTTCTTCGGAGTT3 ' (SEQ ID NO:150).

47. according to the described compositions of claim 43, wherein said B1 chain comprises nucleotide sequence 5 ' GGAUCUUAUUU3 ' (SEQ ID NO:156), wherein said B2 chain comprises nucleotide sequence 5 ' CUUCGGAGTT3 ' (SEQ ID NO:157), and wherein said A chain comprises nucleotide sequence 5 ' CUCCGAAGAAAUAAGAUCCTT3 ' (SEQ ID NO:158).

48. according to the described compositions of claim 43, wherein said B1 chain comprises nucleotide sequence 5 ' GGATCTTATTT3 ' (SEQ ID NO:162), wherein said B2 chain comprises nucleotide sequence 5 ' CTTCGGAGTT3 ' (SEQ ID NO:163), and wherein said A chain comprises nucleotide sequence 5 ' CTCCGAAGAAATAAGATCCTT3 ' (SEQ ID NO:164).

49. according to the described compositions of claim 43, wherein said B1 chain comprises nucleotide sequence 5 ' CTCCGAAGAA3 ' (SEQ ID NO:39), wherein said B2 chain comprises nucleotide sequence 5 ' ATAAGATCCTT3 ' (SEQ ID NO:40), and wherein said A chain comprises nucleotide sequence 5 ' GGATCTTATTTCTTCGGAGTT3 ' (SEQ ID NO:38).

50. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1 B2);

Wherein A:B1 B2 comprises that about altogether 14 base pairs arrive about 24 base pairs altogether;

Wherein A represents sense strand and B1 B2 represents antisense strand;

Wherein A is that about 19 nucleotide are to about 25 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide; With

Wherein the pattern length of B1+B2 is that about 13 nucleotide are to about 23 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

51. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1 B2);

Wherein A:B1 B2 comprises that about altogether 16 base pairs arrive about 22 base pairs altogether;

Wherein A represents sense strand and B1 B2 represents antisense strand;

Wherein A is that about 19 nucleotide are to about 23 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide; With

Wherein the pattern length of B1+B2 is that about 13 nucleotide are to about 23 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

52. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1 B2);

Wherein A:B1 B2 comprises that about altogether 14 base pairs arrive about 24 base pairs altogether;

Wherein A represents antisense strand and B1 B2 represents sense strand;

Wherein A is that about 14 nucleotide are to about 24 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide; With

Wherein the pattern length of B1+B2 is that about 18 nucleotide are to about 24 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

53. a compositions, it comprises:

(a) little inhibition nucleic acid (siRNA) molecule, described siRNA molecule comprises three chain A, B1 and B2 (A:B1 B2);

Wherein A:B1 B2 comprises that about altogether 14 base pairs arrive about 22 base pairs altogether;

Wherein A represents antisense strand and B1 B2 represents sense strand;

Wherein A is that about 16 nucleotide are to about 22 nucleotide;

Wherein B1 and B2 each be about 1 nucleotide independently to about 15 nucleotide;

Wherein the pattern length of B1+B2 is that about 18 nucleotide are to about 22 nucleotide; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

54. a compositions, it comprises:

(a) according to the described siRNA molecule of arbitrary claim among claim 21 and the 50-53, wherein said siRNA molecule reduces the titre of the target virus that is selected from the group of being made up of retrovirus, respiratory syncytial virus, human metapneumovirus, human parainfluenza virus and influenza virus effectively; With

(b) comprise about 5 to about 40 amino acid whose peptides,

Wherein said siRNA molecule conjugates to described peptide.

55. according to the described compositions of arbitrary claim among claim 21 and the 50-53, wherein said peptide comprises the aminoacid sequence that is selected from by the following group of forming: KRRQRRR (SEQ ID NO:1), RQIKIWFQNRRMKWKK (SEQ ID NO:2), DAATATRGRSAASRPTERPRAPARSASRPRRPVD (SEQ ID NO:3), AAVALLPAVLLALLAP (SEQ ID NO:4), AAVLLPVLLPVLLAAP (SEQID NO:5), VTVLALGALAGVGVG (SEQ ID NO:6), GALFLGWLGAAGSTMGA (SEQ ID NO:7), MGLGLHLLVLAAALQGA (SEQ ID NO:8), LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO:9), GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO:10), TPPKKKRKVEDPKKKK (SEQ ID NO:11), RRRRRRR (SEQ ID NO:12), KLALKLALKALKAALKLA (SEQ ID NO:13), GLFGAIAGFIENGWEG (SEQ ID NO:14), FFGAVIGTIALGVATA (SEQID NO:15), FLGFLLGVGSAIASGV (SEQ ID NO:16), GVFVLGFLGFLATAGS (SEQ ID NO:17), GAAIGLAWIPYFGPAA (SEQID NO: 18), ACTCPYCKDSEGRGSGDPGKKKQHICHIQGCGKVYGKTSHLRAHLRWHTGERPFMC (SEQ ID NO: 19), ACTCPNCKDGEKRSGEQGKKKHVCHIPDCGKTFRKTSLLRAHVRLHTGERPFVC (SEQ ID NO:20), ACTCPNCKEGGGRGTNLGKKKQHICHIPGCGKVYGKTSHLRAHLRWHSGERPFVC (SEQ ID NO:21), ACSCPNCREGEGRGSNEPGKKKQHICHIEGCGKVYGKTSHLRAHLRWHTGERPFIC (SEQ ID NO:22), RCTCPNCTNEMSGLPPIVGPDERGRKQHICHIPGCERLYGKASHLKTHLRWHTGER PFLC (SEQ ID NO:23), TCDCPNCQEAERLGPAGVHLRKKNIHSCHIPGCGKVYGKTSHLKAHLRWHTGERPF VC (SEQ ID NO:24), RCTCPNCKAIKHGDRGSQHTHLCSVPGCGKTYKKTSHLRAHLRKHTGDRPFVC (SEQ ID NO:25), PQISLKKKIFFFIFSNFRGDGKSRIHICHLCNKTYGKTSHLRAHLRGHAGNKPFAC (SEQ ID NO:26), WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO:27), GKINLKALAALAKKIL (SEQ ID NO:28), RVIRVWFQNKRCKDKK (SEQID NO:29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO:30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO:31), KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:33), KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ IDNO:37), RKESYSVYVYKVLKQ (SEQ ID NO:41), KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:42), VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:43), AQKKDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:44), KDGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:45), KKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:46), KRSRKESYSVYVYKVLKQ (SEQ ID NO:47), SYSVYVYKVLKQ (SEQID NO:48), VYVYKVLKQ (SEQ ID NO:49), YKVLKQ (SEQ ID NO:50), KVLKQ (SEQ ID NO:51) and KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ (SEQ ID NO:52).

56. a method that is used to reduce the target virus titer said method comprising the steps of:

(a) target gene of the gene silencing of selection siRNA mediation, wherein said target gene is the target viral gene;

(b) design and/or synthetic suitable siRNA molecule are used for the gene silencing of the siRNA mediation of described target viral gene, and each in the wherein said siRNA molecule includes the duplex of breach or otch and wherein said breach or otch and appears in the sense strand or antisense strand of described siRNA duplex;

(c) yoke closes described siRNA to comprising about 5 to about 40 amino acid whose peptides; With

(d) described siRNA molecule is administered to the cell of expressing described target viral gene,

Wherein this siRNA molecular peptide conjugates can specificity in conjunction with corresponding target virus mRNA thereby reduce its expression in cell.

57. one kind is used to reduce the method that endogenous gene is expressed, said method comprising the steps of:

(a) target gene of the gene silencing of selection siRNA mediation, wherein said target gene is an endogenous gene;

(b) the duplex siRNA molecule of design and/or synthetic suitable jagged or otch is used for the gene silencing of the siRNA mediation of described endogenous target gene, and wherein said siRNA molecule includes the duplex of breach or otch and wherein said breach or otch and appears in the sense strand or antisense strand of described siRNA molecule; With

(c) yoke closes described siRNA to comprising about 5 to about 40 amino acid whose peptides; With

(d) described siRNA molecule is administered to the cell of expressing described endogenous target gene,

Wherein said siRNA molecular peptide conjugates energy specificity is in conjunction with corresponding endogenous said target mrna thereby reduce its expression in cell.

58. according to the described compositions of arbitrary claim among the claim 1-13, it is as the medicament of improvement with TNF-α related inflammation.

59. according to the described compositions of claim 58, wherein said inflammation occurs in the arthritis.

60. according to the described compositions of claim 58, wherein said inflammation occurs in the psoriasis.

61. compositions according to claim 1, it is as the medicament of improvement with the influenza virus infections relating.

62. according to the application of the described compositions of arbitrary claim among the claim 1-13 in the medicament of preparation improvement and TNF-α related inflammation.

63. according to the described application of claim 62, wherein said inflammation occurs in the arthritis.

64. according to the described application of claim 62, wherein said inflammation occurs in the psoriasis.

65. the application of compositions according to claim 1 in the medicament of preparation improvement and influenza virus infections relating.

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