CN101410518A

CN101410518A - dsRNA compositions and methods for treating HPV infection

Info

Publication number: CN101410518A
Application number: CNA2007800105410A
Authority: CN
Inventors: J·本松; B·布拉姆利奇; K·菲茨杰拉德; P·丹; H-P·沃恩洛赫尔
Original assignee: Novartis AG
Priority date: 2006-03-24
Filing date: 2007-03-23
Publication date: 2009-04-15

Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for treating human papilloma virus (HPV) infection. The dsRNA comprises an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of an HPV Target gene selected from among HPV E1, HPV E6 and the human E6AP gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by HPV infection and the expression of the E6AP gene using the pharmaceutical composition; and methods for inhibiting the expression of the HPV Target genes in a cell.

Description

DsRNA composition and method that treatment HPV infects

Invention field

The present invention relates to double stranded RNA (dsRNA) and relate to it disturb purposes in the pathologic process that is infected mediation with treatment by human papillomavirus (HPV) at mediate rna, wherein said pathologic process is cervical cancer, anus cancer, precancerous lesion and the Genital warts relevant with HPV for example.

Papilloma virus (PV) is to bring out the no coating dna virus that epithelium hyperplasia venereal disease becomes.Papilloma virus extensively distributes at nature, and quilt cognition in high vertebrates.Virus characterizes in the mankind, ox, rabbit, horse and dog especially.1933, the first routine papilloma virus was described as rabbit papilloma virus (CRPV).From that time, with rabbit papilloma virus and bovine papillomavirus type 1 (BPV-1) experimental prototype as the research papilloma virus.Most of animal teat tumor virus and simple epithelial proliferation venereal disease are in a disguised form closed, and the majority of pathologies of animal is a skin.In the people, identified to surpass 100 types papilloma virus (HPV), and they have been classified: skin epithelium and mucous epithelium (oral mucosa and sexual organ mucous membrane) according to sites of infection.Skin related disease comprises verruca plana, plantar wart etc.The mucous membrane relative disease comprises the larynx papilloma and comprises anus genital diseases (Fields, 1996, Virology, third edition Lippincott--Raven Pub., Philadelphia, the N.Y. of cervical cancer; Bernard, H-U., 2005.J.Clin.Virol.328:S1-S6).

Human papillomavirus (HPV) is that most popular in the world spreading through sex intercourse infected one of disease.It is harmless that most of HPV infect.The HPV of some types causes Genital warts, and these Genital wartss occur with single or multiple lumps at the genital region of masculinity and femininity, and wherein said genital region comprises vagina, uterine neck, vulva (vagina exterior domain), penis and rectum.The people of many infection HPV is asymptomatic.

Most of HPV hypotypes cause benign lesion, but think some hypotypes be high-risk property and can cause more serious pathology, the heteroplasia of uterine cervix and anus for example.Recently, 15 HPV types are divided into high-risk property type (Munoz, people such as N., 2003.N.Engl.J.Med.348 (6): 518-27).These high-risk property hypotypes are that heredity is upward different, and the sequence difference of demonstration＞10% on main viral capsid proteins L1 gene (Bernard, H-U., 2005.J.Clin.Virol.328:S1-S6).

The women who has infected HPV does not often have symptom, and only may just find its pathology after the uterine neck examination.The uterine neck examination uses the Pap test extensively to carry out.Pap test is the Histological assessment that is used to identify the cervical tissue of abnormal cervical cells.(Maryland U.S.A) can determine existence and the special hypotype thereof that HPV infects for Digene, Gaithersburg based on the test example of nucleic acid such as PCR or business-like hybrid capture II technology (HCII) in use.

According to evaluation, unusual cervical cell is classified as has the LSIL (low SIL) (cell (" intracutaneous tumorigenesis-1 on the uterine cervix ") that comprises called after CIN-1) that low risk develops into cancer, or have the HSIL (HSIL) that develops into cancer than high likelihood, comprise the cell of called after CIL-2 and CIL-3.

About 85% the spontaneous degeneration of low pathology, and remaining low pathology remains unchanged or worsens and is the height pathology.If do not treat, expect that about 10% height pathology will change into cancerous tissue.HPV-16 is the most relevant with heteroplasia the most commonly with HPV-18, and is also relevant with heteroplasia although some other HPV transforms hypotype.

Nearest studies show that, the HPV of these high-risk hypotypes of the positive nance's PI of the HIV up to 89%.The HIV positive patient also more may infect the HPV of multiple hypotype simultaneously, and this is relevant with more high-risk heteroplasia progress.

Past, vicennial evidence caused people to accept extensively: it is necessary but non-sufficient that HPV infects cervical cancer.The exist rate of assessment HPV in cervical cancer is 99.7%.Think that there is similar contact in the anus cancer between HPV infection and the dysplastic generation of anus, and think that the situation of anus cancer and cervical cancer is identical.In the research that the HIV negative patient who suffers from the anus cancer is carried out, in 88% anus cancer, found the HPV infection.In the U.S., estimated newly-increased cervical cancer case and 4, the 100 example cervical cancer death of 12,200 examples in 2003, be accompanied by 4,000 examples newly-increased anus carninomatosis example and 500 routine anus cancer death.In in the past 40 years, owing to popularize preventative examination, the sickness rate of cervical cancer decreases, but the sickness rate of anus cancer is increasing.The increase of anus cancer morbidity may part be infected owing to HIV, and this is because the HIV positive patient has higher anus cancer morbidity than general crowd.The sickness rate that the anus cancer has per 100,000 people, 0.9 example in general crowd, and the sickness rate that the anus cancer has per 100,000 people, 35 examples in faggotry crowd, and the sickness rate that in HIV male faggotry crowd, has per 100,000 people 70-100 examples.In fact; because the rising tendency of dysplastic high morbidity of anus and anus cancer in the HIV infected patient, USPHA/IDSA will comprise about being diagnosed as anus heteroplasia patient's treatment policy about the guilding principle for the treatment of the opportunistic infection of HIV positive patient in 2003.

HPV infects does not still have known therapy.Though Genital warts is not even treated usually and can be eliminated yet, and has the methods of treatment to it.Methods of treatment depends on factors such as the size of Genital warts for example and position.Used treatment is Imiquimod emulsion, 20% podophyllin antimitotic solution, 0.5% podophyllotoxin (podofilox) solution, 5% 5 FU 5 fluorouracil emulsion and trichoroacetic acid(TCA).Do not recommend the pregnant woman is used podophyllin or podophyllotoxin because they by skin absorption, and may cause inborn defect.Do not recommend the pregnant woman yet and use 5 FU 5 fluorouracil emulsion.Little Genital warts can remove by freezing (cryosurgery), calcination (electric cautery) or laser therapy with coming physical property.Other is treated nullvalent big wart then must extract by surgical operation.Known Genital warts can recur after carrying out physical removal; In this case, directly alpha-interferon is injected into these warts.Yet alpha-interferon costs an arm and a leg, and uses it can't reduce the recurrence rate of Genital warts.

Therefore, there is effectively this unsatisfied demand of treatment HPV.Surprisingly, found that compound satisfies this demand, and other benefit is provided.

Recently, show that double stranded rna molecule (dsRNA) is to be known as the expression that RNA disturbs the high conservative regulation mechanism blocking gene of (RNAi).WO 99/32619 (people such as Fire) discloses to the purposes of dsRNA inhibition of gene expression in beautiful nematode (C.elegans) of 25 Nucleotide of the youthful and the elderly.Also show, dsRNA comprises that other organism plant is (referring to for example people and WO 99/61631 such as WO 99/53050, Waterhouse, people such as Heifetz), fruit bat is (referring to for example Yang, people such as D, Curr.Biol. (2000) 10:1191-1200) and Mammals (referring to WO 00/44895, Limmer; With DE 10100586.5, people such as Kreutzer) middle degraded target RNA.Now, this natural mechanism has become the development treatment because the focus of the pharmaceutical preparations of the illness that gene unconventionality or unnecessary adjusting cause.

Disclose among the open WO 03/008573 of PCT previous research and development treatment HPV infect the disease that causes, based on the effort of the medicament of nucleic acid.The disclosure has reported that the siRNA of two kinds of target HPV mRNA suppresses the purposes that HPV duplicates in based on the system of cell; At Jiang, people such as M, 2005.N.A.R.33 (18): found relevant open among the e151.

Although marked improvement is arranged, and in the pathologic process of treatment HPV infection mediation, get along with, but still need to suppress the HPV progression of infection and can treat the medicament that infects relevant disease with HPV in the RNAi field.Because this type of medicament must be designed to suppress all high-risk property, show the multifarious HPV hypotype of genotype of extensive degree jointly, so this challenge is severe more.

Summary of the invention

The invention provides by using the reticent HPV of double stranded RNA (dsRNA) to breed necessary genetic expression and treat the solution that infects this problem of relative disease with HPV.E6AP is the conservative gene of the essential human host species of HPV propagation.

The invention provides the double stranded RNA (dsRNA) of inhibition E6AP genetic expression in cell or Mammals and the method for composition and this kind of use dsRNA.The present invention also provides treatment owing to infect pathology illness that the E6AP genetic expression interrelate causes and the disease for example composition and the method for cervical cancer and Genital warts with HPV.DsRNA of the present invention comprise have length be less than 30 Nucleotide, usually a long 19-24 Nucleotide and with the RNA chain (antisense strand) to the basic complementary of small part mRNA transcript zone of E6AP gene.

In one embodiment, the invention provides double stranded RNA (dsRNA) molecule that suppresses E6AP genetic expression.DsRNA comprises at least two sequences complimentary to one another.DsRNA comprises the sense strand that contains first sequence and contains the antisense strand of second sequence.Antisense strand comprises the nucleotide sequence to small part mRNA that is complementary to coding E6AP substantially, and complementary region length is lower than 30 Nucleotide, a long 19-24 Nucleotide usually.After the cells contacting of dsRNA and expression E6AP, at least 40% suppresses E6AP genetic expression.

For example, dsRNA molecule of the present invention can comprise second sequence that table 1 has first sequence of adopted sequence and is selected from table 1 antisense sequences that is selected from of dsRNA.DsRNA molecule of the present invention can comprise naturally occurring Nucleotide or comprise at least one modified Nucleotide, for example the Nucleotide modified of 2 '-O-methyl, comprise the Nucleotide of 5 '-thiophosphoric acid ester group and the terminal nucleotide that is connected with the cholesteric derivative.Alternatively, modified Nucleotide can be selected from: Nucleotide, the morpholino Nucleotide that the Nucleotide (abasic nucleotide), 2 ' of the Nucleotide that the Nucleotide, 2 ' that 2 '-deoxidation-2 '-fluorine is modified-deoxidation is modified, lock Nucleotide (locked nucleotide), dealkalize base-amido modified Nucleotide, 2 '-alkyl is modified, comprise the Nucleotide of phosphoramidate and comprise the Nucleotide of non-natural base.Generally speaking, this type of modified sequence is based on second sequence that table 1 has first sequence of adopted sequence and is selected from table 1 antisense sequences that is selected from of described dsRNA.

In another embodiment, the invention provides the cell that comprises one of dsRNA of the present invention.This cell is generally mammalian cell, for example people's cell.

In another embodiment, the invention provides the pharmaceutical composition that suppresses E6AP genetic expression in organism, usually in people experimenter, it comprises one or more dsRNA of the present invention and pharmaceutically acceptable carrier or delivery vehicle.

In another embodiment, the invention provides and suppress the method that the E6AP gene is expressed in cell, described method comprises the following steps:

(a) introduce double stranded RNA (dsRNA) in cell, wherein said dsRNA comprises at least two sequences complimentary to one another.DsRNA comprises the sense strand that contains first sequence and contains the antisense strand of second sequence.Antisense strand comprises the nucleotide sequence to small part mRNA that is complementary to coding E6AP substantially, and complementary region length is lower than 30 Nucleotide, a long 19-24 Nucleotide usually, and after the cells contacting of wherein said dsRNA and expression E6AP, at least 40% suppresses E6AP genetic expression; With

(b) cell that produces in the step (a) is kept of the degraded of enough time, thereby suppressed the expression of E6AP gene in cell with acquisition E6AP gene mRNA transcript.

In another embodiment, the invention provides for example method of cancer or Genital warts of pathologic process that treatment, prevention or management infect mediation by HPV, described method comprises one or more dsRNA of the present invention to the patient's administering therapeutic significant quantity or the prevention significant quantity of such treatment of needs, prevention or management.

In another embodiment, the invention provides and suppress the carrier that the E6AP gene is expressed in cell, described carrier comprises the effective adjusting sequence that is connected of nucleotide sequence of inventing at least one chain of one of dsRNA with code book.

In another embodiment, the invention provides such cell, described cell comprises and suppress the carrier that the E6AP gene is expressed in cell.Carrier comprises the effective adjusting sequence that is connected of nucleotide sequence of inventing at least one chain of one of dsRNA with code book.

The accompanying drawing summary

Not having figure shows

Detailed Description Of The Invention

The invention provides by using the reticent HPV of double stranded RNA (dsRNA) to breed necessary gene expression and treat and this solution of problem scheme of HPV infection relevant disease. Particularly, the reticent HPV gene E1 of dsRNA of the present invention or E6 or HPV breed necessary human host's conservative gene-people E6AP. At this paper, these genes are referred to as the HPV target gene sometimes.

The invention provides double stranded RNA (dsRNA) and in cell or mammal, use described dsRNA to suppress composition and the method for E1, E6 or E6AP gene expression. The present invention also is provided at and uses the dsRNA treatment by infecting relevant E1, E6 or composition and the method for the caused pathology illness of E6AP gene expression and disease with HPV in the mammal. DsRNA disturbs the sequence-specific degraded of the process instruction mRNA of (RNAi) by being known as RNA.

DsRNA of the present invention comprises to have and is lower than 30 nucleotides, a common long 19-24 nucleotides and substantially be complementary to the RNA chain (antisense strand) in the zone of at least part of HPV said target mrna transcript on the length. The use of these dsRNA is so that relate to the mRNA target degraded of the gene that HPV copies and/or keeps in the mammal. Use is based on cell with based on the test of animal, and the inventor confirms, very these dsRNA of low dosage just can be special and mediate rna i effectively, causes the remarkable inhibition of E1, E6 or E6AP gene expression. Therefore, the inventive method and the composition that comprise these dsRNA can be treated the pathologic process that is infected mediation by HPV for the host factor gene that relates to the HPV life cycle by target.

The description of HPV target-HPV E1 and E6 and people E6AP:

The complex of human host's cell ubiquitin ligase E6AP by itself and viral E6 protein participates in the copying of HPV that HPV especially integrates (non-free) form. E6 is bonded to the protein of many adjusting cell proliferation approach, and often cause its degraded (Chakrabarti, O. and Krishna, S.2003.J.Biosci.28:337-348). E6 and E6AP form complex and come target tumor inhibiting factor p53, thereby degrade (Scheffner, the people such as M, 1990.Cell.63:1129-1136; And Scheffner, M etc., 1993.Cell 75:495-505). By inactivation p53, virus not only stops apoptosis (Chakrabarti and the Krishna of the infected cell of p53 mediation, 2003) and promote viral DNA copy (otherwise copying of viral DNA will be blocked by p53) (Lepik, the people such as D, 1998.J.Virol.72:6822-6831), and virus also is beneficial to tumour generation (Thomas by the control to genome conformity that reduces the p53 mediation, the people such as M, 1999.Oncogene. 18:7690-7700).

E1 and E6 are both at Lippincott-Raven Publishers, Philadelphia, published in 1996 by Bernard N.Fields, the 947-978 page or leaf that the Fundamental Virology that David M.Knipe and Peter M.Howley write is the 3rd edition has carried out quite detailed description by Peter M.Howley in " Papillomaviridae:The Viruses and Their Replication ". The ORF coding DNA of E1 copies the protein of essential 68-76kD. The E1 product of total length is the phosphorylation nucleoprotein in conjunction with the origin of replication among the BPV1 LCR. Show that also E1 is called the total length E2 protein that E2 transcribes trans-activator (E2TA) in conjunction with ATP and in external combination, thus the transcribing of enhanced virus. Also strengthen E1 to the compatibility of dna replication dna starting point with the combination of E2. In HPV-16, E1 has indirect effect to immortalization.

E6 is little alkaline cell transformation protein (for example, HPV16 E6 comprises 151 amino acid), and approximately 16-19kD is positioned in paralinin and the non-nuclear membrane fraction. The E6 gene outcome contains 4 Cys-X-X-Cys motifs, shows potential zinc combination, and it also can be used as nucleic acid binding protein matter. At high-risk property HPV for example among the HPV-16, E6 and E7 protein are that the immortality of squamous cell is essential and sufficient for its host. Show that the E6 gene outcome of high-risk property HPV and p53 form complex, and promote the p53 degraded.

Following detailed description discloses the expression that the composition that how to prepare and use dsRNA and contain dsRNA suppresses the HPV target gene, and treatment is infected the disease that causes and illness for example composition and the method for cervical carcinoma and genital wart by HPV. Pharmaceutical composition of the present invention comprises dsRNA and the pharmaceutically suitable carrier with antisense strand, and wherein said antisense strand comprises length and is lower than 30 nucleotides, a common long 19-24 nucleotides and substantially be complementary to the zone of at least part of rna transcription thing of HPV target gene. One embodiment of the invention adopt the dsRNA more than the different HPV target genes of a kind of randomly target that makes up in pharmaceutical preparation.

Therefore, some aspect of the present invention provides the pharmaceutical composition that comprises dsRNA of the present invention and pharmaceutically suitable carrier, suppresses the method for one or more HPV expression of target gene and make pharmaceutical composition treat the method that is infected the disease that causes by HPV with composition.

I. definition

For convenient, some terms that use in hereinafter furnish an explanation book, embodiment and the claims and the implication of phrase. If there is notable difference in term between the definition that the usage of this specification other parts and its provide in this part, the definition with this part is as the criterion so.

" G ", " C ", " A " and " U " usually represent respectively separately and contain guanine, cytimidine, adenine and uracil as the nucleotides of base. Yet, should be appreciated that term " ribonucleotide " or " nucleotides " also refer to such as the modified nucleotides that hereinafter is described in further detail or the replacing section of substituting. The technical staff knows, and guanine, cytimidine, adenine and uracil can be replaced with other parts and substantially can not be changed the base pairing characteristic of oligonucleotides, and wherein said oligonucleotides comprises the nucleotides with this kind replacing section. For example, be not restricted ground, comprise inosine and can form base-pair with the nucleotides that contains adenine, cytimidine or uracil as the nucleotides of its base. Therefore, the nucleotides that contains uracil, guanine or adenine can be replaced with for example containing the nucleotides of inosine in nucleotide sequence of the present invention. The sequence that comprises this type of replacing section is embodiment of the present invention.

As used herein, " E6AP " refers to ubiquitin protein ligase E3A (ube3A is also referred to as E6 associated protein or E6AP) gene or protein.GenBank accession number NM_130838.1, NM_130839.1 and NM_000462.2 provide the people mRNA sequence of the E6AP of the different isoforms of representative.

As used herein, " E1 " refers to the E1 gene (GenBank accession number NC_001526,865-2813 position Nucleotide) of human papillomavirus Class1 6 (HPV16).As used herein, " E6 " refers to the E6 gene (GenBank accession number NC_001526,65-559 position Nucleotide) of human papillomavirus Class1 6 (HPV16).Many variants of E1 and E6 gene are disclosed.Unless clearly get rid of in the literary composition, this paper be intended to by use " E1 " and " E6 " comprise these and future disclosed E1 and E6 genetic mutation.

As used herein, " target sequence " refers to the sequential portion at the nucleotide sequence of the mRNA molecule of the transcription stage formation of one of HPV target gene, and it is included as the mRNA of the RNA processed products of primary transcription product.

As used herein, term " chain that comprises sequence " refers to the oligonucleotide that comprises a succession of Nucleotide, and wherein said a succession of Nucleotide is described by the sequence of using the standard nucleotides nomenclature to refer to.

As used herein, and unless otherwise indicated, otherwise term " complementary " is when being used for describing first nucleotide sequence with respect to second nucleotide sequence, it refers to the oligonucleotide that comprises first nucleotide sequence or polynucleotide under certain condition with oligonucleotide that comprises second nucleotide sequence or multi-nucleotide hybrid and form the ability of duplex structure, and this point is understood by the technician.This type of condition can be a stringent condition for example, and wherein said stringent condition can comprise: 50 ℃ of 400mMNaCl, 40mM PIPES pH6.4,1mM EDTA or 70 ℃ 12-16 hour, washing then.Also can use other condition, the physiology correlated condition that for example may run in vivo.The technician can be according to the condition setting that finally should be used for determining the complementary test of the most suitable two sequences of hybridization Nucleotide.

This comprises the base pairing that oligonucleotide or polynucleotide that comprise first nucleotide sequence and the oligonucleotide that comprises second nucleotide sequence or polynucleotide list at first and second nucleotides sequences of total length.At this paper, this type of sequence can be called each other " complementary fully ".Yet, at this paper, when wherein first sequence is called with second sequence " basic complementary ", these two sequences can be complementary fully, perhaps they are in final using when keeping the hybridization ability under the maximally related condition with it, can forming one or morely after hybridization, but common no more than 4,3 or 2 base mismatch are right.Yet two oligonucleotide being designed to after hybridization to form one or more strands when outstanding, according to the mensuration of complementarity, this type of is outstanding not to be considered as mispairing.For example, comprise the oligonucleotide of long 21 Nucleotide and the dsRNA of the oligonucleotide of another long 23 Nucleotide, wherein long oligonucleotide comprises and sequence than 21 Nucleotide of the complete complementary of short oligonucleotide,, still can be called " complementary fully " for purpose of the present invention.

As used herein, " complementary " sequence also can comprise non-Watson-Crick base pair and/or the base pair that is formed by non-natural and modified Nucleotide, perhaps form by above-mentioned base pair fully, as long as described sequence realizes its demand to the hybridization ability.

At this paper, term " complementary ", " complementary fully " and " basic complementary " can be used between dsRNA sense strand and the antisense strand or the base pairing between dsRNA antisense strand and the target sequence, understand in the purposes of this paper as it.

As used herein, refer to basic complementary polynucleotide with the polynucleotide of " complementary substantially " of messenger RNA(mRNA) (mRNA) with the purpose mRNA (E6AP for example encodes) of sequential portion to small part.For example, if non-the interruptions part complementation substantially of oligonucleotide sequence and the mRNA of coding E6AP, so oligonucleotide at least with part E6AP mRNA complementation.

As used herein, term " double-stranded RNA " or " dsRNA " refer to the ribonucleic acid molecule complex body of the duplex structure with the nucleic acid chains that comprises two antiparallel and complementary substantially (definition as mentioned).Two chains that form the duplex structure can be the different pieces of big RNA molecule, or the RNA molecule that separates.When being that RNA separately divides the period of the day from 11 p.m. to 1 a.m, this type of dsRNA is commonly referred to siRNA (" short RNA interfering ") in the literature.When two chains are a more macromolecular part, and therefore continual a succession of Nucleotide connection between the 5 ' end of 3 ' terminal and another chain of a chain by forming the duplex structure, the RNA chain of connection is called " hairpin loop ", " little hairpin RNA " or " shRNA ".When two chains are not continual a succession of Nucleotide and when covalently bound, syndeton is called " joint " between the 5 ' end of 3 ' terminal and another chain of a chain by forming the duplex structure.The RNA chain can have the Nucleotide of identical or different quantity.The maximum number of base pair is to have deducted the Nucleotide number of the shortest outstanding dsRNA chain arbitrarily that exists in the duplex structure.Except the duplex structure, it is outstanding that dsRNA can comprise one or more Nucleotide.In addition, used as this specification sheets, " dsRNA " can comprise the chemically modified to bonding, end group, cap and conjugate moiety between ribonucleotide, nucleosides, comprising modifying in the essence at a plurality of Nucleotide place and comprising that this paper discloses or all types of modification known in the art.For the purpose of this specification sheets and claim, " dsRNA " comprises any this type of modification of using in the molecule of siRNA type.

As used herein, " Nucleotide is outstanding " refers to the unpaired Nucleotide of giving prominence to from the duplex structure of dsRNA when 3 ' end of a chain of dsRNA extends to the 5 ' end that exceeds another chain, and perhaps vice versa.The described end that " flat " or " flush end " is illustrated in dsRNA does not have unpaired Nucleotide, does not promptly have Nucleotide outstanding." flat end " dsRNA is for all being double-stranded dsRNA on its total length, promptly two ends at molecule all do not have Nucleotide outstanding.Should be understood that whether have outstanding or whether be flat when terminal measuring siRNA, do not consider 3 ' terminal or 5 ' the terminal chemical cap that connects or the chemical part of non-nucleotide in conjunction with siRNA.

Term " antisense strand " refers to the chain with the basic complementary of target sequence zone of comprising of dsRNA.As used herein, term " complementary zone " refers on the antisense strand and the sequence basic complementary of the target sequence zone of this paper definition for example.When complementary region and target sequence are not exclusively complementary, the tolerance mispairing in zone endways, and if present, zone or for example in the zone in 5 ' and/or 3 ' terminal 6,5,4,3 or 2 Nucleotide endways usually.

As used herein, term " sense strand " refers to comprising and the regional chain in complementary zone substantially of antisense strand of dsRNA.

Understand ground as those skilled in the art, when referring to dsRNA, " introducing in cell " expression promotes cellular uptake or absorption.The absorption of dsRNA or picked-up can be by carrying out from the cell processes of main diffusion or active or by complementary reagent or device.The implication of this term is not limited to external cell, and when cell during for the part of the organism that lives, dsRNA also can " introduce cell ".In this case, cytotropic introducing comprises sending to organism.For example, dsRNA can be injected into tissue site or systemic administration carries out sending in the body.Comprise methods known in the art for example electroporation and lipofection in external cytotropic introducing.

At this paper, as long as term " silence " and " suppressing to express " relate to the HPV target gene, it refers to the target gene expression to small part inhibition HPV so, this minimizing by the mRNA amount that the HPV target gene is transcribed shows, wherein said mRNA can be from having transcribed the HPV target gene, and carried out first cell or first group of cellular segregation that treatment is suppressed the HPV target gene expression, and wherein said mRNA amount is reduced to and basic identical with first cell or first group of cell, but the result that second cell of so treating or second group of cell (control cells) are compared.The inhibition degree is expression in the following manner usually:

Alternatively, the inhibition degree can provide in the mode that the parameter relevant with HPV target gene functional transcription descends, and wherein said parameter is the amount or for example apoptotic cell number of demonstration particular phenotype by HPV target gene encoded protein matter of emiocytosis for example.In principle, HPV target gene silence can and can detect by the test of any appropriate in the cell of any expression target (constitutive expression or by the genome project technological expression).Yet, when needs with reference to determine specific dsRNA whether suppress to a certain extent the HPV target gene expression, and so be contained in when of the present invention, hereinafter the test that provides of embodiment will be with for referencial use.

For example, in some cases, suppress about at least 20%, 25%, 35% or 50% E6AP genetic expression by using double chain oligonucleotide of the present invention.In some embodiments, suppress about at least 60%, 70% or 80% E6AP gene by using double chain oligonucleotide of the present invention.In some embodiments, suppress about at least 85%, 90% or 95% E6AP gene by using double chain oligonucleotide of the present invention.Table 2 provides the value of the transcripting suppressioning action that obtains in a large number in vitro test, the multiple E6AP dsRNA molecule of multiple concentration is used in wherein said test.Similarly, table 6 provides the value of a large amount of E1 transcripting suppressioning actions; And table 8 provides the value of a large amount of E6 transcripting suppressioning actions.

As used in the literary composition that infects at this paper HPV, term " treatment ", " therapy " etc. refer to alleviates or slows down the pathologic process that HPV infects mediation.This description comprises to be used therapeutical agent prevention of the present invention or prevents that HPV from infecting and alleviation HPV infects symptom or the illness that causes.In the context of the invention, relating under hereinafter described any other illness situation of (being different from the pathologic process that HPV infects mediation) expressions such as term " treatment ", " therapy " alleviation or alleviate the symptom of at least a and described disease-related or slow down or reverse the process of described disease.

As used herein, phrase " treatment significant quantity " and " prevention significant quantity " refer to the quantity that the treatment benefit is provided in the manifest symptom of the pathologic process of mediation or the pathologic process that HPV infects mediation is infected in treatment, prevention or management by HPV.Treating effective specified quantitative can be determined easily by the general doctor, and depend on factor well known in the art and difference, wherein said factor is for example infected type, patient's the medical history of the pathologic process of mediation and age, is infected the stage of pathologic process and the using of other disease-resistant reason agent of mediation by HPV by HPV.

As used herein, " pharmaceutical composition " comprises dsRNA and pharmaceutically acceptable carrier of pharmacy effective dose.As used herein, " pharmacy effective dose ", " treatment significant quantity " or simple " significant quantity " refer to the dsRNA dosage of pharmacy effect, result of treatment or the preventive effect of effective generation expection.For example, if when the minimizing that has at least 25% in the measurable parameter relevant, just think that given clinical treatment is that effectively the treatment significant quantity for the treatment of the medicine of this disease or illness so is to make above-mentioned parameter be reduced by at least 25% required amount with disease or illness.

Term " pharmaceutically acceptable carrier " refers to the carrier of administering therapeutic agent.Examples of such carriers includes but not limited to salt solution, buffer saline, glucose, water, glycerol, ethanol and combination thereof.Cell culture medium clearly got rid of in term.For the medicine of dosage forms for oral administration, pharmaceutically acceptable carrier includes but not limited to pharmaceutically acceptable vehicle, for example inert diluent, cracking agent, tackiness agent, lubricant, sweeting agent, seasonings, tinting material and sanitas.Suitable inert diluent comprises the phosphoric acid salt and the lactose of carbonate, sodium and the calcium of sodium and calcium, and W-Gum and alginic acid then are suitable cracking agents.Tackiness agent can comprise starch and gelatin, and lubricant (if present) is generally Magnesium Stearate, stearic acid or talcum powder.If necessary, tablet can postpone in GI absorption with the material dressing of for example glyceryl monostearate or distearin.

As used herein, " through cell transformed " is for having introduced the cell of expressing the carrier of dsRNA molecule.

II. double stranded RNA (dsRNA)

In one embodiment, the invention provides the double stranded ribonucleic acid molecule (dsRNA) that in cell or Mammals, suppresses the HPV expression of target gene, the antisense strand that wherein said dsRNA comprises comprise with when expressing the HPV target gene, form on length, be lower than 30 Nucleotide, the zone of a long 19-24 Nucleotide usually to small part mRNA complementation and complementary region, and after the cells contacting of wherein said dsRNA and the described HPV target gene of expression, at least 10%, 25% or 40% suppresses described HPV expression of target gene.

DsRNA comprises two abundant complementations so that hybridization forms the RNA chain of duplex structure.The chain (antisense strand) of dsRNA comprises the complementary substantially and usually complete complementary complementary region with target sequence, the mRNA sequence that described target sequence forms when coming comfortable HPV target sequence to express, another chain (sense strand) comprise with the antisense strand complementation so that the hybridization and form the zone of duplex structure when combining under proper condition of two chains.Generally speaking, the duplex scantling length between 15-30 the base pair, be more typically between 18-25 the base pair, further more normally between 19-24 base pair, the most normally between 19-21 base pair.Similarly, and the complementary region length of target sequence is between 15-30 Nucleotide, more generally between 18-25 Nucleotide, further more normally between 19-24 Nucleotide, the most normally between 19-21 Nucleotide.It is outstanding that dsRNA of the present invention can further comprise one or more strand Nucleotide.DsRNA can be as hereinafter further synthesizing by standard method well known in the art with discussing, and wherein said method is for example by using for example Biosearch, Applied Biosystems, the commercial automatic dna synthesizer of Inc..In preferred embodiments, HPV target gene behaviour H6AP gene.In specific embodiment, the antisense strand of dsRNA comprises and is selected from table 1 and the chain of adopted sequence is arranged and be selected from second sequence of the antisense sequences of table 1.The alternative antisense reagent in other place of the target sequence that target table 1 provides can use the E6AP sequence of target sequence and flank to determine easily.

In further embodiment, dsRNA comprises at least one nucleotide sequence that is selected from the sequence that table 1 provides.In other embodiments, dsRNA comprises at least two sequences that are selected from this group, wherein another sequence complementation in sequence at least two sequences and at least two sequences, and the mRNA sequence complementation substantially that produces during with E6AP genetic expression of a sequence at least two sequences.Generally speaking, dsRNA comprises two oligonucleotide, and one of them oligonucleotide is described as the sense strand in the table 1, and second oligonucleotide is described as the antisense strand in the table 1.Table 1 provides the duplex title of each preferred dsRNA and serial ID number.

In further embodiment, dsRNA comprises at least a sequence, the named double-stranded dsRNA that table 5 (E1 dsRNA) or table 7 (E6 dsRNA) provide that be selected from.

The technician knows: comprise between 20 to 23, particularly the dsRNA of the duplex structure of 21 base pairs especially effectively (people such as Elbashir, EMBO 2001,20:6877-6888) in disturbing bringing out RNA.Yet other people finds that shorter or longer dsRNA is also effective.In the above-described embodiment, by the character of the oligonucleotide sequence that provides in table 1, table 5 or table 7, dsRNA of the present invention can comprise that at least one length is minimum to be the chain of 21nt.Can reasonable expectation, the shorter dsRNA that comprise a sequence in table 1, table 5 or the table 7, only deducts several Nucleotide at one or two end can have with above-mentioned dsRAN and compares similar effects.Therefore, the present invention's partial sequence and restraining effect that its energy force rate that suppresses the HPV expression of target gene comprises the dsRNA of complete sequence in FACS test as herein described or other test of having considered to comprise at least 15,16,17,18,19,20 or more a plurality of continuous nucleotides in one of the sequence of table 1, table 5 or table 7 is hanged down and is no more than 5,10,15,20,25 or 30% dsRNA.Other dsRNA that cuts in the target sequence that table 1, table 5 or table 7 provide can use the reference sequences and the target sequence that provide to generate easily.

In addition, the RNAi reagent that provides in table 1, table 5 and table 7 has been determined the site in HPV said target mrna separately, and wherein said said target mrna is to the cutting sensitivity based on RNAi.So, the present invention further comprises the RNAi reagent in the sequence of one of target reagent of the present invention target.As used herein, if optional position in the cutting of second kind of RNAi reagent and the first kind of RNAi reagent antisense strand complementary mRNA, second kind of RNAi reagent just is called the sequence of first kind of RNAi reagent of target so.At least 15 continuous nucleotides of one of sequence that this second kind of reagent is provided by table 1, table 5 or table 7 are usually formed, wherein said Nucleotide and the extra nucleotide sequence coupling that is derived from the selected sequence adjacent domain in the HPV target gene.For example, 6 Nucleotide of vicinity of last 15 Nucleotide (deducting the AA sequence of interpolation) of SEQ ID NO:1 and target E6AP gene make up the strand reagent of 21 Nucleotide that one of sequence of providing based on table 1 is provided.

DsRNA of the present invention can contain the one or more mispairing to target sequence.In preferred embodiments, dsRNA of the present invention contains and is no more than 3 mispairing.If the antisense strand of dsRNA contains the mispairing to target sequence, the preferred no fix in mispairing zone is at the center of complementary region so.If the antisense strand of dsRNA contains the mispairing to target sequence, mispairing is preferably limited to 5 Nucleotide of arbitrary end so, for example 5,4,3,2 or 1 Nucleotide of 5 ' of complementary region or 3 ' end.For example, for the dsRNA chain of 23 Nucleotide of the regional complementarity of HPV target gene, dsRNA does not contain any mispairing usually in 13 Nucleotide at center.Method of the present invention can be used to measure the mismatched dsRNA that comprises target sequence and whether effectively suppress the HPV target gene expression.It is important considering to have the validity of mismatched dsRNA in suppressing the HPV expression of target gene, if especially known particular complementary zone in the HPV target gene is in virus (if E1 or E6) or the sequence difference that has polymorphism in crowd (for E6AP).

In one embodiment, it is outstanding that at least one end of dsRNA has the strand Nucleotide of 1-4, common 1 or 2 Nucleotide.Having the outstanding dsRNA of at least one Nucleotide has unexpected highly inhibited than flat terminal dsRNA.In addition, present inventors find, only have the outstanding interferon activity of having strengthened dsRNA of a Nucleotide, and do not influence its resistance to overturning.Verified, it is especially stable and effectively in vivo and in various kinds of cell, cell culture medium, blood and the serum only to have an outstanding dsRNA.Generally speaking, the outstanding 3 ' end that is positioned at antisense strand of strand is perhaps alternatively at 3 ' end of sense strand.DsRNA also can have flat terminal, and it is positioned at 5 ' end of antisense strand usually.This type of dsRNA has the stability of improvement and suppresses active, therefore allows to use low dosage, and promptly every day, every kilogram of experimenter's body weight was less than 5mg.Generally speaking, the antisense strand of dsRNA has Nucleotide at 3 ' end to be given prominence to, and 5 ' end is flush end.In another embodiment, usefulness nucleosides thiophosphatephosphorothioate substitutes the one or more Nucleotide in giving prominence to.

Again in another embodiment, dsRNA is carried out chemically modified and come enhanced stability.Nucleic acid of the present invention can synthesize and/or modification by the method that this area has been set up, wherein said method is " Current protocols in nucleic acid chemistry " for example, Beaucage, people such as S.L (writing), John Wiley ﹠amp; Sons, Inc., New York, NY, the method described in the USA, described document is incorporated herein by reference herein.Chemically modified can include but not limited to 2 ' to modify, the modification on other site of the sugar of oligonucleotide or base, the non-natural base is introduced oligonucleotide chain, covalently bound to part or chemical part, and with alternative key phosphoric acid ester bond between thiophosphatephosphorothioate replacement Nucleotide for example.Can adopt more than this type of a kind of modification.

The chemistry connection of two chains that separate of dsRNA can realize by multiple well-known technology, for example by introducing covalent linkage, ionic linkage or hydrogen bond, hydrophobic interaction, Van der Waals interaction or accumulative facies mutual effect, pass through metallic ion coordination or passing through to use purine analogue.Generally speaking, the chemical group that can be used for modifying dsRNA includes but not limited to methylenum coeruleum; The double functional group is generally two-(2-chloroethyl) amine; N-ethanoyl-N '-(to the glyoxylyl benzoyl) cystamine; 4-thiouracil and psoralene.In one embodiment, joint is the hexaethylene glycol joint.In this case, dsRNA produces by solid-phase synthesis, and introduces the hexaethylene glycol joint according to standard method (for example, Williams, D.J and K.B.Hall, Biochem. (1996) 35:14665-14670).In specific embodiment, 5 ' end of antisense strand and 3 ' end of sense strand carry out chemistry by the hexaethylene glycol joint and are connected.In another embodiment, at least one Nucleotide of dsRNA comprises thiophosphoric acid ester group or phosphorodithioic acid ester group.The chemical bond of dsRNA end generally forms by the triple helix key.Table 1 provides the present invention the example of modified RNAi reagent.

Again in another embodiment, can modify the degraded that Nucleotide in two strands one or two chains prevented or suppressed cell enzyme activity, wherein said enzyme is such as but not limited to some nuclease.The active technology of inhibition cellular enzymes degraded nucleic acid known in the art, comprising but be not limited to 2 '-amido modified, 2 '-aminosugar is modified, 2 '-F is sugar-modified, 2 '-F modifications, sugar-modified, the uncharged backbone modifications of 2 '-alkyl, morpholino are modified, the modification of 2 '-O-methyl and phosphoramidate be (referring to for example, Wagner, Nat.Med. (1995) 1:1116-8).Therefore, at least one 2 '-oh group of the last Nucleotide of dsRNA with chemical group, use 2 ' usually-amino or 2 '-methyl group substitutes.And, can modify at least one Nucleotide and form lock Nucleotide.This lock Nucleotide contains the methylene bridge that connects ribose 2 '-O and ribose 4 '-C.Contain lock Nucleotide oligonucleotide at Koshkin, people such as A.A, Tetrahedron (1998), 54:3607-3630) and Obika, people such as S, Tetrahedron Lett. (1998) is described in 39:5401-5404).With lock Nucleotide introduce oligonucleotide improved to a certain extent to the affinity of complementary sequence and improved melting temperature(Tm) (Braasch, D.A and D.R.Corey, Chem.Biol. (2001), 8:1-7).

Part is conjugated to dsRNA can be promoted its intracellular absorption and promote its target in particular organization or promote it by for example vaginal epithelial cell picked-up of the cell of particular type.In some cases, hydrophobic ligand is conjugated to the direct infiltration that dsRNA promotes cytolemma.Alternatively, the part that is conjugated to dsRNA is the substrate that is used for receptor-mediated endocytosis.These methods have been used to promote the Premeabilisation of cells of antisense oligonucleotide and dsRNA reagent.For example, cholesterol has been conjugated to multiple antisense oligonucleotide, thereby has produced than the more effective in fact compound of its unconjugated analogue.Referring to M.Manoharan Antisense ﹠amp; Nucleic Acid DrugDevelopment 2002,12,103.Other lipophilic compound that is conjugated to oligonucleotide comprises 1-pyrene butyric acid, 1,3-two-O-(hexadecyl) glycerol and menthol.An example that is used for the part of receptor-mediated endocytosis is a folic acid.Folic acid enters cell by folacin receptor mediated endocytosis.The dsRNA compound that has folic acid effectively transports cell by folacin receptor mediated endocytosis.Lee and co-worker's report thereof are connected to 3 ' of oligonucleotide-end with folic acid and cause the picked-up of oligonucleotide in the cell to increase by 8 times.Li，S.；Deshmukh，H.M.；Huang，L.Pharm.Res.1998，15，1540。Other part that is conjugated to oligonucleotide comprises polyoxyethylene glycol, sugar bunch, linking agent, porphyrin conjugate and send peptide.

In some cases, puting together of positively charged ion part and oligonucleotide causes resistance that nuclease is improved.The representative instance of positively charged ion part is propyl ammonium and dimethyl propyl ammonium.What is interesting is that according to reports, when the positively charged ion part was dispersed in the oligonucleotide, antisense oligonucleotide kept its high binding affinity to mRNA.Referring to M.Manoharan Antisense ﹠amp; Nucleic Acid DrugDevelopment 2002,12,103 and reference wherein.

The dsRNA that part of the present invention is puted together can for example be connected in link molecule dsRNA and go up and obtain by making the dsRNA of the reactive functional group that apparatus dangles.This reactive oligonucleotide can be directly with commercial part, synthesize part or have the part reaction that is connected connection portion thereon with kinds of protect group.In some preferred embodiments, method of the present invention by use suitably puted together part, and the nucleoside monomers that can further be connected to the solid phase support material promote that dsRNA's that part is puted together is synthetic.According to the certain preferred embodiments of the inventive method, part-nucleosides conjugate that examples of such optional is connected to the solid phase support material reacts with the connection portion that is positioned at nucleosides or oligonucleotide 5 ' position by selected serum binding partner and prepares.In some cases, the dsRNA with the aralkyl part that is connected to dsRNA 3 '-end is by preparing by the long-chain aminoalkyl group is at first covalently bound the monomer structure unit to the controlled pore glass support.Then, Nucleotide is bonded to the monomer structure unit of puting together with solid support by the solid phase synthesis technique of standard.The monomer structure unit can be nucleosides or other organic compound with the solid-phase synthesis compatibility.

The dsRNA that is used for conjugate of the present invention can make things convenient for and be prepared by well-known solid phase synthesis technique routinely.This kind synthetic equipment sold by some sellers, comprising for example Applied Biosystems (Foster City, CA).Additionally or alternatively, can adopt this kind synthetic any other method that is used for known in the art.Also known use similar techniques prepares other oligonucleotide, for example thiophosphatephosphorothioate and alkyl derivative.

Synthetic instruction about the oligonucleotide of specific modification can be found in following United States Patent (USP): about puting together the oligonucleotide of polyamines, see U.S. Patent number 5,138,045 and 5,218,105; About being used to prepare the monomer of oligonucleotide, see U.S. Patent number 5,212,295 with chiral phosphorus key; About the oligonucleotide of the modified main chain of tool, see U.S. Patent number 5,378,825 and 5,541,307; About the oligonucleotide of modified main chain and the preparation by reductive coupling thereof, see U.S. Patent number 5,386,023; About modified nucleic acid base and synthetic method thereof, see U.S. Patent number 5,457,191 based on 3-deazapurine loop systems; About modified nucleic acid base, see U.S. Patent number 5,459,255 based on the N-2 substituted purin; Have the method for the oligonucleotide of chiral phosphorus key about preparation, see U.S. Patent number 5,521,302; U.S. Patent number 5,539,082 about peptide nucleic acid(PNA); About having the oligonucleotide of beta-lactam main chain, see U.S. Patent number 5,554,746; About the method and the material of synthetic oligonucleotide, see U.S. Patent number 5,571,902; About having the nucleosides of alkylthio group, see U.S. Patent number 5,578,718, wherein said this type of group can as with the joint of the other parts of any a plurality of positions that are connected nucleosides; About the oligonucleotide of phosphorothioate bond, see U.S. Patent number 5,587,361 and 5,599,797 with high chiral purity; About preparing the method for 2 '-O-alkyl guanosine and related compound, see U.S. Patent number 5,506,351, comprising the 2,6-diaminopurine compound; About having the oligonucleotide of N-2 alternate purine, see U.S. Patent number 5,587,469; About having the oligonucleotide of 3-deazapurine, see U.S. Patent number 5,587,470; About having puted together 4 '-demethyl nucleoside analog, see U.S. Patent number 5,223,168 and 5,608,046 the two; About the oligonucleotide analogs of modified main chain, see U.S. Patent number 5,602,240 and 5,610,289; About Synthetic 2 '-method of fluoro-oligonucleotide, especially see U.S. Patent number 6,262,241 and 5,459,255.

Be connected with among the dsRNA and part-molecule that the part of sequence-specific nucleosides of the present invention puts together at tool, oligonucleotide and oligonucleoside can utilize standard nucleotides or nucleosides precursor or contain the Nucleotide of connection portion or nucleosides conjugate precursor, the non-nucleosides part that contained the part-Nucleotide or the nucleosides conjugate precursor of ligand molecular or contained structural unit are assembled on suitable dna synthesizer.

When using the Nucleotide contained the connection portion-conjugate precursor, generally finish the synthetic of nucleosides that sequence-specific connects, and ligand molecular and connection portion are reacted form the oligonucleotide that part is puted together subsequently.Contain for example oligonucleotide conjugate existing description (referring to people such as Manoharan, PCT applies for WO93/07883) before of steroid, VITAMIN, lipid and reporter molecules of multiple molecule.In preferred embodiments, the nucleosides of oligonucleotide of the present invention or connection uses from the phosphoramidite (phosphoramidite) of part-nucleosides conjugate and the commercial standard phosphoramidite and the non-standard phosphoramidite of conventional use in oligonucleotide is synthetic by the automatization synthesizer and synthesizes.

In the nucleosides of oligonucleotide, mix 2 '-O-methyl, 2 '-O-ethyl, 2 '-O-propyl group, 2 '-O-allyl group, 2 '-O-aminoalkyl or 2 '-deoxidation-2 '-fluoro group and give oligonucleotide enhanced hybridization characteristic.Further, the oligonucleotide that contains the thiophosphatephosphorothioate main chain has the enhanced nuclease stability.Therefore, the nucleosides of the connection that the present invention is functionalized can comprise that perhaps the two strengthens by comprising the thiophosphatephosphorothioate main chain or comprising 2 '-O-methyl, 2 '-O-ethyl, 2 '-O-propyl group, 2 '-O-aminoalkyl, 2 '-O-allyl group or 2 '-deoxidation-2 '-fluoro group.Oligonucleotides-modified summary tabulations more known in the art can for example found among the PCT application WO 200370918.

In some embodiments, the present invention uses the dna synthesizer preparation at the functionalized nucleotide sequences that 5 '-end has amino group, and subsequently with the active ester derivative reaction of selected part.Active ester derivative is well known to those skilled in the art.Representational active ester comprises N-hydroxy-succinamide ester, tetrafluoro phenolic ester, pentafluranol ester and pentachloro-phenolic ester.The reaction of amino group and active ester generates oligonucleotide, and wherein selected part is connected to 5 '-position by linking group.5 '-terminal amino group can utilize 5 '-amido modified thing C6 reagent preparation.In one embodiment, ligand molecular can be puted together in 5 ' of oligonucleotide-position by using part-nucleoside phosphoramidites, and wherein said part directly or indirectly is connected in 5 '-oh group by joint.What this type of part-nucleoside phosphoramidites was used in the automatization synthesis step usually was provided at the oligonucleotide that part that 5 '-end has part is puted together the most afterwards.

The example of key or main chain comprises between the nucleosides of modifying, for example thiophosphatephosphorothioate, the chirality thiophosphatephosphorothioate, phosphorodithioate, phosphotriester, the aminoalkyl phosphotriester, methylphosphonate and other phosphonate ester that comprises 3 '-alkylene phosphonic acids ester and chiral phosphonate, phosphinate, the phosphoramidate that comprises 3 '-amino phosphoramidate and aminoalkyl phosphoramidate, thiocarbonyl group phosphoramidate (thionophosphoramidates), thiocarbonyl group phosphonate ester (thionoalkylphosphonates), thiocarbonyl group alkyl phosphotriester (thionoalkylphosphotriesters) and have the borine phosphoric acid ester (boranophosphates) of conventional 3 '-5 ' key, the analogue that 2 ' of these materials-5 ' connect, and those have opposite polarity, wherein adjacent nucleosides unit is to being 3 '-5 ' to 5 '-3 ' or 2 '-5 ' to 5 '-2 ' material that connects.Also comprise multiple salt, mixing salt and free acid form.

Relate to the above-mentioned representative United States Patent (USP) that contains the phosphorus atom key of preparation and include but not limited to U.S. Patent number 3,687,808,4,469,863,4,476,301,5,023,243,5,177,196,5,188,897,5,264,423,5,276,019,5,278,302,5,286,717,5,321,131,5,399,676,5,405,939,5,453,496,5,455,233,5,466,677,5,476,925,5,519,126,5,536,821,5,541,306,5,550,111,5,563,253,5,571,799,5,587,361,5,625,050 and 5,697,248, wherein each patent all is incorporated herein by reference herein.

The example that does not wherein comprise bonding between the nucleosides of modification of phosphorus atom or main chain (being oligonucleoside) has the main chain by bonding forms between bonding or one or more short chain heteroatomss or heterocyclic sugar between key, blended heteroatoms and alkyl or cycloalkyl sugar between short-chain alkyl or cycloalkyl sugar.These comprise and have the morpholino key (part is formed by the sugar moieties of nucleosides), siloxane main chain, thioether, sulfoxide and sulfone main chain, oxygen methylene oxygen base (formacetyl) and sulfenyl methylene radical oxygen base (thioformacetyl) main chain, methylene radical oxygen methylene oxygen base and sulfonium methylide methylene oxygen base main chain, contain the main chain of alkene; Sulfamate backbone, methylene imido grpup and methylene diazanyl main chain, sulphonate and sulfonamide backbone; Amide backbone and other have the main chain of blended N, O, S and CH2 component part.

Relating to the representative United States Patent (USP) for preparing above-mentioned oligonucleotide includes but not limited to: U.S. Patent number 5,034,506,5,166,315,5,185,444,5,214,134,5,216,141,5,235,033,5,264,562,5,264,564,5,405,938,5,434,257,5,466,677,5,470,967,5,489,677,5,541,307,5,561,225,5,596,086,5,602,240,5,610,289,5,602,240,5,608,046,5,610,289,5,618,704,5,623,070,5,663,312,5,633,360,5,677,437 and 5,677,439, wherein each patent is all quoted as a reference herein.

In some cases, oligonucleotide can be modified by non-ligand groups.Many non-ligand moleculars are conjugated to oligonucleotide strengthening activity, cell distribution or the cellular uptake of oligonucleotide, and have in scientific literature and carry out the method that this type of is puted together.This type of non-ligand moiety comprises the lipid part, cholesterol (people such as Letsinger, Proc.Natl.Acad.Sci.USA, 1989 for example, 86:6553), cholic acid (people such as Manoharan, Bioorg.Med.Chem.Lett., 1994,4:1053), thioether, hexyl-S-trityl mercaptan (tritylthiol) (people such as Manoharan for example, Ann.N.Y.Acad.Sci., 1992,660:306; People such as Manoharan, Bioorg.Med.Chem.Let., 1993,3:2765), sulfo-cholesterol (people such as Oberhauser, Nucl.Acids Res., 1992,20:533), aliphatic chain, for example dodecanediol or undecyl residue (people such as Saison-Behmoaras, EMBO J., 1991,10:111; People such as Kabanov, FEBS Lett., 1990,259:327; People such as Svinarchuk, Biochimie, 1993,75:49), phosphatide, for example two-hexadecyl-racemize-glycerol or triethyl ammonium 1,2-two-O-hexadecyl-racemize-glycerol-3-H-phosphonic acid ester (people such as Manoharan, Tetrahedron Lett., 1995,36:3651; Shea etc., Nucl.Acids Res., 1990,18:3777), polyamines or polyglycol chain (people such as Manoharan, Nucleosides ﹠amp; Nucleotides, 1995,14:969) or adamantane acetic acid (people such as Manoharan, Tetrahedron Lett., 1995,36:3651), palmityl part (people such as Mishra, Biochem.Biophys.Acta, 1995,1264:229) or stearylamine or hexyl amino-carbonyl-oxycholesterol part (people such as Crooke, J.Pharmacol.Exp.Ther., 1996,277:923).The representative United States Patent (USP) of this class oligonucleotide conjugate of instruction preparation is above being listed.The general scheme of puting together relates to and synthesizes the oligonucleotide that has amino joint in one or more positions of sequence.Use suitable coupling agent or activator with amino group and the molecular reaction of puting together subsequently.Conjugation reaction can be carried out after the solution phase with still puting together in the oligonucleotide of solid support or at the cutting oligonucleotide.Usually provide purified conjugate by HPLC purification of oligonucleotides conjugate.Especially preferentially use cholesterol conjugate, because this molecule can increase the target to vagina epithelium histocyte (site that HPV infects).

The disclosure has been described the multiple embodiments that is used for reticent HPV target gene, also therefore treats the dsRNA of HPV associated conditions.Though can take various ways to design particular therapeutic agent, some functional characters will be distinguished preferred dsRNA from other dsRNA.Particularly, detect for example good serum stability of all features that those skilled in the art can measure, high-effect, the preferred dsRNA of the present invention is not identified in induction of immunity reaction and good medicine sample behavior.In some cases, be not that all these function aspects all appear among the preferred dsRNA.But those skilled in the art can optimize these variablees and other variable is selected the preferred compound of the present invention.

Though many nucleotide modifications all are possible, present inventors have identified pharmacology, immunology that remarkable improvement is provided and the chemically modified pattern of finally treating benefit.Table 9 has been showed the chemically modified pattern that is preferred for the double-stranded dsRNA that lists in table 1 of the present invention, table 5 and the table 7.During these are modified some are also set forth in table 3.

Table 9

Chemically modified series	The change that sense strand (5 '-3 ') is carried out	The change that antisense strand (5 '-3 ') is carried out
Chemically modified series	The change that sense strand (5 '-3 ') is carried out	The change that antisense strand (5 '-3 ') is carried out	1 (at the single thiophosphatephosphorothioate of two chain ends)	dTsdT	dTsdT
2 (at the single thiophosphatephosphorothioate of two chain ends, and modification of 2 ' OMe sense strand of all pyrimidines and the 2 ' Ome modification that is all U of A thereafter, be thereafter that 2 ' Ome of all C of A modifies)	DTsdT, all Py of 2 ' OMe@	dTsdT，2′OMe@uA， cA		dTsdT	dTsdT
	DTsdT, all Py of 2 ' OMe@	dTsdT，2′OMe@uA， cA	3 (at the single thiophosphatephosphorothioate of two chain ends, and 2 ' OMe sense strand of all pyrimidines is modified, modify with the 2 ' Ome that is all U of A on antisense strand thereafter, thereafter 2 ' the Ome that is all C of A modifies, thereafter 2 ' the Ome that is all U of G modifies, and is thereafter that 2 ' Ome of all U of U modifies)	DTsdT, all Py of 2 ' OMe@	dTsdT，2′OMe@uA，c A，uG，uU
4 (except adding is conjugated to the cholesterol of sense strand, identical) with 1	Chol(″exo″)	dTsdT(″exo″)		DTsdT, all Py of 2 ' OMe@	dTsdT，2′OMe@uA，c A，uG，uU
	Chol(″exo″)	dTsdT(″exo″)	5 (except the cholesterol that is conjugated to sense strand, identical) with 2	Chol(″endo″)	dTsdT，2′OMe@uA， cA
6 (except the cholesterol that is conjugated to sense strand, identical) with 3	Chol(″endo″)	dTsdT，2′OMe@uA，c A，uG，uU		Chol(″endo″)	dTsdT，2′OMe@uA， cA

The carrier of coding RNA i reagent

DsRNA of the present invention also can be expressed in vivo by intracellular recombinant viral vector.Recombinant viral vector of the present invention comprises the sequence of code book invention dsRNA and is used to express any suitable promotor of dsRNA sequence.Suitable promotor comprises for example U6 or H1 RNA pol III promoter sequence, and cytomegalovirus promoter.Select other suitable promotor to belong to art technology.Recombinant viral vector of the present invention also can be included in particular organization or be used to express inducing or adjustable promotor of dsRNA in the environment in particular cell.Send dsRNA of the present invention to cell in the use recombinant viral vector body and will carry out more detailed discussion hereinafter.

DsRNA of the present invention can be expressed as two complementary RNA molecules that separate by recombinant viral vector, perhaps is expressed as the single rna molecule of two complementary region of tool.

Can use any virus vector that can accept the encoding sequence of dsRNA molecule to be expressed, for example derived from the carriers of adenovirus (AV), adeno associated virus (AAV), retrovirus (for example slow virus (LV), rhabdovirus, murine leukemia virus), simplexvirus etc.The tropism of virus vector can be by with other viral envelope protein or other surface antigen pseudotyping carrier or modify by replacing different viral capsid proteins as required.

For example, lentiviral vectors of the present invention can come pseudotyping with the surface protein of blister oral membrane inflammation virus (VSV), rabies virus, Ai Baila virus, mokola virus etc.Can AAV preparing carriers of the present invention be become the different cells of target by carrier being transformed into the different capsid protein serotypes of expression.For example, the AAV carrier of expression serotype 2 capsids is called AAV2/2 on serotype 2 genomes.This serotype 2 capsid genes in the AAV2/2 carrier can be replaced to serotype 5 capsid genes and produce the AAV2/5 carrier.The technology of the AAV carrier of the different capsid protein serotypes of construction expression is the technology in this area, referring to, people (2002) such as Rabinowitz J E for example, J Virol76:791-801, it is incorporated herein by reference with its integral body herein.

The nucleotide sequence that selection is applicable to recombinant viral vector of the present invention, will express dsRNA inserts the method for carrier and all is art technology with the method that virus vector is delivered to the purpose cell.Referring to, Dornburg R (1995) for example, Gene Therap.2:301-310; Eglitis M A (1988), Biotechniques 6:608-614; Miller A D (1990), Hum Gene Therap.1:5-14; Anderson W F (1998), people such as Nature 392:25-30 and Rubinson D A, Nat.Genet.33:401-406, described document its integral body herein are incorporated herein by reference.

Preferred virus vector is the carrier derived from AV and AAV.In particularly preferred embodiments, dsRNA of the present invention is two complementary single stranded RNA molecules that separate by the reorganization AAV vector expression that comprises U6 for example or H1 RNA promotor or cytomegalovirus (CMV) promotor.

Express dsRNA of the present invention suitable AV carrier, make up the method for reorganization AV carrier and carrier sent method into target cell people (2002) such as Xia H, be described among the Nat.Biotech.20:1006-1010.

Express the suitable AAV carrier of dsRNA of the present invention, make up the method for reorganization AV carrier, and carrier sent method into target cell people (1987) such as Samulski R, J.Virol.61:3096-3101, people (1996) such as Fisher K J, J.Virol, 70:520-532, people (1989) such as Samulski R, J.Virol.63:3822-3826, U.S. Patent number 5,252,479, U.S. Patent number 5,139,941, be described among international patent application no WO 94/13788 and the international patent application no WO 93/24641, all documents are incorporated herein by reference with its integral body herein.

III. the pharmaceutical composition that comprises dsRNA

In one embodiment, the invention provides and comprise dsRNA as described herein and the pharmaceutical composition of pharmaceutically acceptable carrier.The pharmaceutical composition that comprises dsRNA is used for the treatment of and HPV target gene expression or active relevant disease or illness, and for example HPV infects the pathologic process of mediation.This type of pharmaceutical composition carries out preparation according to delivery modality.Example is preparation for topical application in uterine neck or sends the composition of systemic administration through parenteral.

Pharmaceutical composition of the present invention is used the dosage that enough suppresses the HPV expression of target gene.Present inventors determine that because the effect of its improvement, the composition that comprises dsRNA of the present invention can be used with surprising low dosage.The dsRNA dosage of every kilogram of experimenter's body weight 5mg enough suppressed or stoped the HPV expression of target gene every day, and in the situation of wart, uterine neck or anus treatment, above-mentioned dosage can directly apply to infected tissue.

Generally speaking, the suitable dosage of dsRNA is in the scope of 0.01 to 5.0 milligram of every kilogram of experimenter's body weight every day, usually in the scope of every kg body weight 1 microgram to 1 milligram every day.Pharmaceutical composition can be used once every day, perhaps dsRNA can the appropriate intervals of every day use twice, three times or more frequently sub-doses or even sustained release preparation by vaginal jellies inject continuously or send.In the sort of situation, the dsRNA that contains in each sub-doses must be correspondingly littler so that reach total dosage every day.Also can mix dose unit and send some days, for example use to be provided at the conventional extended release preparation that continues to discharge dsRNA in some days.Extended release preparation is to know in this area, and is particularly useful for the vagina delivery of agents, for example can use with reagent of the present invention.In this embodiment, dose unit contains corresponding a plurality of every day of dosage.

The technician understands, and some factors can influence required dosage and the time course of effective treatment experimenter, comprising but be not limited to the seriousness of disease or illness, former treatment, experimenter's holistic health and/or other disease of age and existence.In addition, the combination treatment experimenter with the treatment significant quantity comprises single therapy or serial therapy.Can use ordinary method or carry out the assessment of transformation period in the effective dose of each dsRNA that the present invention includes and the body, as described in other place of this paper based on the body build-in test that uses suitable animal model.

Present inventors recognize, owing to multiple reason, may need to use simultaneously more than a kind of dsRNA of the present invention and treat the HPV infection, and wherein said reason comprises the genotypic otherness of HPV.In one embodiment, the combination of selection dsRNA comes the HPV genotype with minimum dsRNA complex mixture target maximum range.Expectation comprises the dosage that contains each dsRNA as described herein more than the pharmaceutical composition of the present invention of one type dsRNA.

The combination of dsRNA can provide in single forms of pharmaceutical compositions together.Alternatively, the combination of dsRNA can provide to divide other formulation, and in this case, other formulation of described branch can be used simultaneously or at different time, and may be by different modes.Therefore, the present invention has considered to comprise the pharmaceutical composition of the dsRNA combination of the present invention of expection, and it has also considered to be intended to the pharmaceutical composition of single dsRNA of kind that the part as assembled scheme provides.Therefore in the later case, the invention of combination treatment is application process but not the composition of entity.

Produced big quantity research various human class disease is for example infected the pathologic process of mediation by HPV mouse model in the genetic progress of mouse.This class model is used for body build-in test dsRNA, and is used for determining the treatment significant quantity.

Can use any method to come to the administration that contains the cell that has infected HPV dsRNA of the present invention.For example, use can be partial (for example vagina, endermic etc.), per os or parenteral (for example, by in subcutaneous, the ventricle, intramuscular or peritoneal injection, perhaps by intravenous drip).Using can be (for example by injection) fast, perhaps can take place for some time (for example by slow infusion or use slow delivery formulations).

Usually, when treatment has the Mammals of the cell that has infected HPV, topical application dsRNA molecule in vagina gel or emulsion.For example, with or can directly be applied to uterine neck, rectum road or the HPV injury region of Genital warts for example partly without the dsRNA of Liposomal formulation.For topical application, the dsRNA molecular preparation can be gone into composition, described composition is for example aseptic and the aqueous solution of bacterium is arranged, at conventional solvent non-aqueous solution or the solution in liquid state or solid oil matrix in the alcohol for example.This type of solution also can contain buffer reagent, thinner and other suitable additive.Be used for topical application of compositions and can become transdermal patch, ointment, lotion, emulsion, gelifying agent, drops, suppository, sprays, liquid and form of powder by preparation.Gelifying agent and emulsion can use polymer known in the art and saturatingization agent to prepare.Gelifying agent or the emulsion that contains dsRNA and relevant vehicle can use diaphragm, contraceptive diaphragm, have by condom, gloves etc. and be applied to uterine neck.Can add conventional pharmaceutical carrier, water, powder or oleaginous base, thickening material etc.

For in parenteral, the sheath or intraventricular using, the dsRNA molecular preparation can be gone into composition, sterile aqueous solution for example, it also can contain buffer reagent, thinner and other suitable additive (for example penetration enhancer, carrier compound and other pharmaceutically acceptable carrier).

In addition, can use non-viral method to the administration dsRNA that contains the cell that has infected HPV, wherein said method is U.S. Patent number 6,271 for example, biology described in 359 or abiology method.Abiological sending can be realized by several different methods, comprising but be not limited to (1) and load liposome with dsRNA acidic molecular provided herein and (2) compound dsRNA molecule and lipid or liposome to form nucleic acid-lipid or nucleic acid-liposome compound.Liposome can comprise cationic lipid and the neutral fat that is generally used for the in-vitro transfection cell.Cationic lipid can with the compound liposome that forms of the nucleic acid of negative charge.The example of cationic-liposome includes but not limited to lipofectin, lipofectamine, lipofectace, DOTAP (1; 2-two oleoyls-3-trimethyl ammonium propane), DOTMA (N-[1; 2 (2; 3-two oil base oxygen bases) propyl group]-N; N; the N-trimethyl ammonium chloride), DOSPA (2; 3-two oily acyloxy-N-[2-(spermine carboxylic acid amides) ethyl]-N; N-dimethyl-1-propyl ammonium), DOGS (two octadecyl acid amides glycyl spermine) and DC-chol (3; [N-N ', N '-dimethyl ethylene diamine)-formamyl] cholesterol).

The process that forms liposome is well known.Liposome composition can for example be formed by phosphatidylcholine, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidylcholine, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL or DOPE.A large amount of lipophilic reagent are commercial, comprising Lipofectin.RTM. (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene.TM. (Qiagen, Valencia, Calif.).In addition, the cationic lipid that the systemic delivery method can commodity in useization for example DDAB or DOTAP is optimized, and wherein said every kind of cationic lipid can for example DOPE or cholesterol be mixed with neutral fat.In some cases, those liposomes that can use people (Nature Biotechnology, 15:647-652 (1997)) such as Templeton for example to describe.In other embodiments, can use polycation for example polymine comes in the perfect aspect and the sending of exsomatize (ex vivo) people such as (, J.Am Soc.Nephrol.7:1728 (1996)) Boletta.Can be about the extraneous information of using liposome delivery nucleic acid at U.S. Patent number 6,271,359, the open WO 96/40964 of PCT and Morrissey, people such as D, 2005.Nat Biotechnol.23 (8): find among the 1002-7.

Other non-viral method to the administration dsRNA molecule that contains the cell that has infected HPV comprises (except liposome) also based on the delivery system of cationic lipid, for example lipoplexes and nano-emulsion.Extraly, can use the polymkeric substance delivery system of condensation (is the DNA polymer composite body, perhaps " polyplexes "), comprising but be not limited to chitosan, poly-(L-Methionin) (PLL), polymine (PEI), dendrimer (dendeimers) (for example polyamidoamines (PANAM)) dendrimer) and ethylenediamine polyoxyethylene polyoxypropylene block polymer (poloxamine).Extraly, can use the polymkeric substance delivery system of non-condensation, comprising but be not limited to polyoxyethylene polyoxypropylene block copolymer (poloxamer), gelatin, PLGA (polylactic-co-glycolic acid), PVP (polyvinylpyrrolidone) and PVA (polyvinyl alcohol).

Above-mentioned send or the process of application technique is well known.For example, the polymkeric substance delivery system of condensation is by playing a role with the negatively charged ion dna molecular is compound easily; For example, poly-(L-Methionin) (PLL) plays a role by taking the photograph in forming with the interactional positive charge complex body of negative charge cell surface, also carrying out fast subsequently.

Biology is sent and can be passed through accomplished in many ways, comprising but be not limited to use virus vector.For example, can use virus vector (for example, adenovirus and herpesvirus vector) that dsRNA is delivered to skin cells and cervical cell.The Protocols in Molecular Biology that can use standard is incorporated into being used for to one of many different virus carriers of cell nucleic acid delivery of previous research and development with one or more dsRNA provided herein.The carrier of these gained can be used for by for example infecting one or more dsRNA being delivered to cell.

DsRNA of the present invention can preparation in pharmaceutically acceptable carrier or thinner." pharmaceutically acceptable carrier " (this paper is also referred to as " vehicle ") is acceptable solvent, suspension agent or any other pharmacy inert support.Pharmaceutically acceptable carrier can be liquid or solid-state, and can select to provide anticipated volume, consistence and other suitable transportation and chemical property with the application method of plan.General pharmaceutically acceptable carrier comprises (by way of example but be not limited to): water, salt brine solution, tackiness agent (for example, polyvinylpyrrolidone or Vltra tears), weighting material (for example lactose and other carbohydrate, gelatin or calcium sulfate), lubricant (for example starch, polyoxyethylene glycol or sodium-acetate), disintegrating agent (for example starch or Explotab) and wetting agent (for example sodium lauryl sulphate).

In addition, the dsRNA preparation of target HPV target gene can be entered contain with other molecule, molecular structure or nucleic acid mixture mix, wrap by, put together or the composition of associating dsRNA.For example, the dsRNA combination of agents thing that contains one or more targets E6AP gene can contain other therapeutical agent, for example anti-inflammatory drug (for example, NSAID (non-steroidal anti-inflammatory drug) and glucocorticosteroid) and antiviral (for example ribavirin (ribivirin), vidarabine (vidarabine), aciclovir (acyclovir) and ganciclovir (ganciclovir)).In some embodiments, composition contains the associating of one or more dsRNA and keratolytic agent (keratolytic agent), and wherein said dsRNA has and HPV target gene complementary sequence.Keratolytic agent is for separating or loosen the reagent of horny layer of epidermis.The example of keratolytic agent includes but not limited to Whitfield's ointment.U.S. Patent number 5,543 provides other example in 417.Can use the keratolytic agent of effective dose to strengthen the infiltration of dsRNA, for example infiltrate through and organize for example skin.For example, the keratolytic agent amount that can infiltrate through wart with the dsRNA that allows to be applied to Genital warts is used.

The toxicity of this compounds and therapeutic efficiency can be measured by standard drug method in cell culture or laboratory animal, for example measure LD50 (to 50% lethal amount of colony) and ED50 (effectively measuring in 50% colony).Dosage rate between toxic action and the therapeutic action is a therapeutic index, and it can be expressed as the ratio of LD50/ED50.The preferred compound that shows high therapeutic index.

The data that obtain from cell culture test and zooscopy can be used for the preparation of the dosage range of human purposes.Usually in the scope of circulation composition, it includes minimum or does not have toxic ED50 the dosage of the present composition.Dosage can change according to the formulation that adopts and the route of administration of use in this scope.For any compound that uses in the inventive method, the treatment significant quantity can be tested with cell culture at first and be assessed.Dosage can be prepared the polypeptide product of realizing compound or target sequence as required in animal model circulating plasma concentration range (for example, realize that peptide concentration reduces), wherein said concentration range is included in the IC50 (promptly reaching the concentration of the maximum restraining effect one half test-compound of symptom) that measures in the cell culture.This type of information can be used for more accurately being determined at the useful dosage of philtrum.Level in blood plasma can for example be passed through high effective liquid chromatography for measuring.

DsRNA of the present invention is except independent or multiple using as discussed above, and it can be infected the medicament of pathologic process of mediation by HPV co-administered with other known effective treatment.In any situation, the administration doctor can be based on using standard effect known in the art or as described herein to measure application dosage and the time course that observed result adjusts dsRNA.

The associating of dsRNA can be used in vitro and in vivo and identify that preferably the list kind same procedure that dsRNA adopted is tested.This type of associating can be selected based on the information biology basis fully, wherein selects to provide the siRNA of the genotypic minimal number of the widest scope of covering.Alternatively, can be based on being used in the external or body that the single dsRNA of kind compositions and methods carries out assessment and selecting this type of associating according to as herein described.The preferred assay method of associating of test dsRNA is (people (2005) Journal Vir.79 (14) such as Hengstermann for example: 9296 in HPV16 male cancerous cell line; With SiHa or the Caski described in people (2003) the Oncogene 22:5938 such as Butz) or at people (1995) Journal Vir.69 (5) such as for example Jeon: the phenotype result that the HPV target of assessment siRNA mediation strikes low (knock down) in the 2989 described organotypic culture systems.

Present inventors have identified that some can be used for the treatment of the preferred associating of the dsRNA of HPV infection.With the most frequently used term, dsRNA unites the dsRNA that is selected from table 1, table 3, table 5 and table 7 that comprises more than a kind of.Therefore, the present invention has considered 2,3,4,5 or the more kinds of purposes of dsRNA duplex in combination therapy that is selected from table 1, table 3, table 5 and table 7.In principle, the dsRNA of preferred minimal number simplifies the treatment product.This impels selects the harmful of cover-most quantity or the genotypic dsRNA of the deleterious HPV of possibility, and can confirm that really the combination of selecting need not to cover this type of all HPV genotype.

Following dsRNA especially is fit to associating

From E1:ND-9072, ND-9142, ND-9092, ND-9162, ND-9097, ND-9167, ND-9066, ND-9123, AL-DP-8082, AL-DP-8095;

From E6:ND-8903, ND-8991, ND-8914, ND-9002, ND-8906, ND-8994, ND-8943, ND-9031, ND-9032, ND-8920, ND-8952, ND-8951, ND-9008, ND-9040, ND-9039, AL-DP-7783, AL-DP-7784;

From E6AP:AL-DP-7365, AL-DP-7371, AL-DP-7499, AL-DP-7545, AL-DP-7492, AL-DP-7473, AL-DP-7478, AL-DP-7554, AL-DP-7514, AL-DP-7397, ND-9300.

The method of the disease that causes is infected in treatment by HPV

Method and composition as herein described can be used for the treatment of disease and the illness that is caused by human papillomavirus, and wherein said disease and illness may be the results of clinical or subclinical parillomarvirus infections.This type of disease and illness (this paper is sometimes referred to as " HPV associated conditions " or " being infected the pathologic process of mediation by HPV ") comprise, for example the epithelium malignant tumour, skin carcinoma (non-melanoma or melanoma), the anus genital malignant tumor is cervical cancer for example, the precancerous lesion (comprising LSIL or HSIL cervical tissue) that HPV is relevant, the anus knurl, malignant change, benign lesion, papilloma, papillo-adenocystoma, papilloma neuropathy (papilloma neuropathicum), papillomatosis, skin and mucous membrane papilloma, condyloma, the pathology illness that fibroblastoma is relevant with papilloma virus with other.

For example, composition as herein described can be used for the treatment of the wart that HPV causes.This type of wart comprises, for example verruca vulgaris (verruca vulgaris), for example palm, sole of the foot flesh and periungual wart; Verruca plana and verruca filiformis; Anus, oral cavity, pharynx, larynx and tongue papilloma; Venereal wart condyloma acuminata (pointed condyloma) also is known as Genital warts (for example, penis, vulva, vagina and uterine neck wart), is one of phenomenon the most serious during HPV infects.HPV DNA can form in (CIN I-III) at all other cervical intraepithelial neoplasias of level and find, and the HPV type of specific hypotype can find in the cancer original position of uterine neck.Therefore, think that the women who suffers from the Genital warts that contains specific HPV type has the high-risk property that cervical cancer takes place.

The common disease relevant with parillomarvirus infections is optimum cutaneous wart or verruca vulgaris.Verruca vulgaris contains HPV Class1,2,3,4 or 10 usually.Other comprises by illness that papilloma virus causes, larynx papilloma for example, and it is the benign epithelial tumor of larynx.Usually, two kinds of papillomavirus type HPV-6 are the most relevant with the larynx papilloma with HPV-11.Composition as herein described can be used for the treatment of these diseases and illness.

Composition described herein also can be used for the treatment of epidermodysplasia verruciformis (EV), and this is a kind of rare inherited disease that is characterized by the dispersivity verruca plana, shows as little erythema.

In addition, composition as herein described can be used for the treatment of and results from the wherein HPV of cell transformation and be the pathology of its cause of disease, for example treats cervical cancer.

Composition as herein described also can be used for the treatment of heteroplasia, for example penis, vulva, uterine neck, vagina, mouth, the anus that HPV brings out and swallow heteroplasia, and the cancer of treatment the HPV cancer, for example penis, vulva, uterine neck, vagina, anus, oral cavity, pharynx and head and the neck that bring out.

It is to comprise the specific dsRNA that unites with for example any conventional chemotherapy agent of another anti-cancer chemotherapeutic agents that the present invention also can use.The associating of particular combination agent and this type of other medicament can be strengthened the chemotherapy scheme.Skilled doctor knows that many chemotherapy schemes can mix method of the present invention.Can use any chemotherapeutics, comprising alkylating agent, antimetabolite, hormone and antagonist, radio isotope and natural product.For example, compound of the present invention can with microbiotic for example Zorubicin and other anthracene nucleus class analogue, mustargen for example endoxan, pyrimidine analogue for example 5 FU 5 fluorouracil, cis-platinum, hydroxyl urea, taxol and natural and synthetic derivative etc. are used.As another example, for example comprise gonad-stimulating hormone dependency and gonad-stimulating hormone not in the situation of the mammary cancer of dependent cell in mixed tumor, compound can be used with Leuprolide or goserelin (the synthetic peptide analogs of LH-RH).Other anti-knurl scheme comprises with another therapeutic modality uses tetracycline compound, for example surgical operation, the radiation etc. of wherein said another therapeutic modality, and this paper is also referred to as " complementary anticancer mode ".Therefore, method of the present invention can have the conventional scheme that reduces side effect and strengthen the effect of effect with this type of and uses.

Further alternatively, can adopt the dsRNA of target E6AP to treat illness on neuroscience and the study of behaviour.By identify the E6AP sudden change in the patient who suffers from angel's syndrome (Angelman syndrome), E6AP has related to the illness on neuroscience and the study of behaviour.Angel's syndrome (AS) be characterized by backwardness, language defect, exceedingly laugh at, the characteristic neurobehavioral illness (Hitchins of epileptic seizures, ataxia and characteristic EEG pattern, people such as M.P, 2004.AmJ Med Genet is (2) A.125: 167-72).By inference, therapeutic purpose also do not lie in and bring out this type of illness, but as observed in many hereditary defectes, E6AP has keying action in neuroscience and study of behaviour illness, this evidence also shows, this target spot has multiple effect and may be the suitable target of other disease in human pathology, wherein said disease belongs to the kind that reticent E6AP then compensates other biological chemistry defective or disease.As used herein, " E6AP associated conditions " comprises illness and other neuroscience and the study of behaviour illness that above-mentioned HPV is relevant.

The method that suppresses E6AP genetic expression

In addition, on the other hand, the invention provides the method that in Mammals, suppresses E6AP genetic expression.Method comprises that composition in administration table 1 of the present invention is so that reticent target E6AP expression of gene.Because specificity of its height, this type of dsRNA of the present invention is the RNA of target target E6AP gene (initial or through processing) specifically.Use this type of dsRNA to suppress that the composition of these E6AP genetic expressions and method can other carries out as described in local as this paper.

In one embodiment, method comprises uses the composition that comprises dsRNA, and wherein said dsRNA comprises the nucleotide sequence to small part rna transcription thing that is complementary to mammiferous E6AP gene to be treated.When organism to be treated is a for example man-hour of Mammals, composition can be used by any method known in the art, comprising but be not limited to per os or parenteral approach, comprising intravenous, intramuscular, subcutaneous, using through (aerosol) skin, air flue, intranasal, rectum, vagina with partial (comprising oral cavity and hypogloeeis).In preferred embodiments, composition is used by part/vaginal application or by intravenous infusion or injection.

Unless otherwise defined, otherwise all technology used herein and scientific terminology all have the identical meanings as those skilled in the art's common sense.Though in practice of the present invention or can use and methods described herein and materials similar or suitable method and material in the test, have hereinafter described appropriate means and material.Mentioned in this article all are open, patent application, patent and other reference all are incorporated herein by reference with its integral body.Under controversial situation, comprise that with this specification sheets definition is as the criterion.In addition, material, method and embodiment are only with explaining, not as restriction.

Embodiment

The gene step of E6AP gene moves

Carry out siRNA and design the siRNA that identifies target human ubiquitin protein ligase enzyme E3A (ube3A is also referred to as E6AP).Use the mRNA sequence (NM_130838.1, NM_130839.1, NM_000462.2) of the different isoforms of representative E6AP.

Use the ClustalW multiple ratio of BioEdit software to function (Thompson J.D. to the somebody E6AP of institute isoform, Deng the people, Nucleic Acids Res.1994,22:4673), identified that mRNA sequence NM_130838.1 is the shortest sequence, and confirm the sequence conservation of the 5-4491 position (terminal position) of reference sequences, the latter is for all E6AP isoforms of target are required effectively.

Identify all possible plyability 19mer of leap E6AP reference sequences NM_130838.1 (representing siRNA sense strand sequence), produced 4473 19mer candidate sequences.Jointly, these candidate's target sequences cover the joint location of 5 ' UTR, coding region and 3 ' UTR structural domain and these structural domains of E6AP mRNA.

For classification from candidate storehouse and select siRNA, with the interactional expection potential of nothing to do with target (target potential (off-target potential) partially) as value parameter.The siRNA that will have low inclined to one side target potential is defined as preferably, and thinks that it is more special in vivo.

Inclined to one side target potential in order to predict that siRNA is special, carry out following hypothesis:

1) complementarity of (from 5 ' to 3 ' counting) (seed region (seed region)) and target gene may be enough (people such as Jackson AL, Nat Biotechnol.2003 June for the interaction of that chain and the mRNA that transcribes from target gene and downward modulation subsequently in the 2-9 position of chain; 21 (6): 635-7)

2) the 1st of every chain the and the 19th interacts irrelevant with inclined to one side target

3) seed region may more have contribution to inclined to one side target potential than the other parts of sequence

4) cleavage site of chain zone the 10th and the 11st (counting from 5 ' to 3 ') may more have contribution to inclined to one side target potential than the sequence (non-seed region) of cleavage site 3 ' side, but not as seed region

5) can when considering hypothesis 1-4, calculate inclined to one side target score value based on the complementarity of siRNA chain-ordering and gene order and the position of mispairing

6) supposition is modified by the inherence of introducing and has been abolished the sense strand activity potentially, and then only the inclined to one side target potential of antisense strand is relevant

7) the inclined to one side target potential of siRNA can infer from the gene (best inclined to one side target gene) that shows highest homology according to our standard, so it can be represented with the inclined to one side target score value of each gene

In order to identify the inclined to one side target gene of potential, the antisense sequences of 19mer is carried out the homology search to the obtainable people mRNA of public sequence.For this purpose, people RefSeq database is carried out fastA (version 3 .4) search with candidate's antisense sequences of all 19mer.Use Perl script (Perl script) to produce antisense sequences (perl script 2) from candidate's 19mer sequence.FastA search execution parameter/value is right-g 30-f 30-L-i-H, so that consider to cross over the homology of 19mer total length and make output format be fit to the former this analysis that goes on foot down.Search produces the potential inclined to one side target gene list of candidate siRNA.

In addition, to be worth E 15000 and be applied to the fastA search parameter, so that enter database with what be same as 19mer sense strand sequence more than 8 continuous nucleoside bases (nucleobases), this very likely is transferred to the fastA output file and shows the homology of 19mer total length (referring to hypothesis 1) simultaneously.

In order to identify best inclined to one side target gene and inclined to one side target score value thereof, the fastA output file is analyzed.For the inclined to one side target gene of each potential, taken passages following inclined to one side target character to each 19mer list entries:

Mispairing number in seed zone

Mispairing number in non-seed zone

Mispairing number in the cleavage site district

The following calculating of inclined to one side target score value to each inclined to one side target gene:

(seed mispairing number * 10)+(cleavage site mispairing number * 1.2)+non-seed mispairing number

The 19mer sequence of each input has been taken passages minimum inclined to one side target score value, and and then it has been write output file, thereby the inclined to one side target that produces all siRNA of the corresponding 19mer sequence of importing divides value list.

In order to produce classification, with inclined to one side target score value input results table to siRNA.At last all siRNA are carried out sort in descending order according to target score value partially, and the sequence that will contain in a row more than the tract of 3 G excludes screening.

Selection has also been synthesized the siRNA (table 1) of 156 inclined to one side target score value＞=3.

DsRNA is synthetic

Reagent source

When the not specifically shown reagent source of this paper, can obtain to be applied to this reagent of molecular biological quality/purity standard from any molecular biology reagent supply and marketing merchant.

SiRNA is synthetic

Single stranded RNA produces with 1 micromolar scale by solid phase synthesis, wherein use Expedite8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG,

, Proligo Biochemie GmbH, Hamburg, Germany) and as solid support.The RNA of RNA and contain 2 '-O-methyl nucleotide adopt respectively corresponding phosphoramidite and 2 '-O-methyl phosphoramidite produces (Proligo BiochemieGmbH, Hamburg, Germany) by solid phase synthesis.These structural units use the nucleoside phosphoramidites chemistry of standard for example as Current protocols in nucleic acid chemistry, Beaucage, people such as S.L. (writing), John Wiley ﹠amp; Sons, Inc., New York, NY is described in the USA. and mix selected site in the sequence of oligoribonucleotide chain.Phosphorothioate bond is by (UK) replacement of the solution in acetonitrile (1%) iodine oxidizing agent solution is introduced for ChruachemLtd, Glasgow with Beaucage reagent.Further auxiliary reagent obtains from Mallinckrodt Baker (Griesheim, Germany).

The going of rough oligoribonucleotide protected with purifying and implemented by anionresin HPLC according to the method for having set up.Output and concentration absorb to determine (DU 640B, Beckman Coulter GmbH, Unterschlei β heim, Germany) by using the UV of each RNA solution of spectrophotometric determination at wavelength 260nm place.Double-stranded RNA passes through at annealing buffer (20mM sodium phosphate, pH6.8; 100mM sodium-chlor) mix equimolar complementary strand solution in, 85-90 ℃ of heating 3 minutes and be cooled to room temperature and surpass 3-4 hour and produce in water-bath.Annealed RNA solution is stored in-20 ℃ until use.

SiRNA (this paper is called-Chol-3 ') for synthetic 3 '-cholesterol-put together uses the solid support of suitably modifying to synthesize RNA.The solid support of modifying is prepared as follows:

2-azepine butane-1,4-diethyl dicarboxylate AA

4.7M aqueous sodium hydroxide solution (50mL) is joined ice-cold glycine ethyl ester hydrochloride (32.19g, 0.23mole) solution in water (50mL) through stirring.Then, add ethyl propenoate (23.1g, 0.23 mole), and finish until confirming to react by TLC at the stirring at room mixture.After 19 hours, with methylene dichloride (3 * 100mL) separation solutions.Organic layer is with anhydrous sodium sulfate drying and filter and evaporate.Distillation residue provide AA (28.8g, 61%).

3-{ ethoxy carbonyl methyl-[6-(9H-fluorenes-9-ylmethoxy carbonyl-amino)-caproyl]-amino }-ethyl propionate AB

(9.12g 25.83mmol) is dissolved in the methylene dichloride (50mL), and with ice-cooled with Fmoc-6-amino-caproic acid.0 ℃ in solution, add DIC (3.25g, 3.99mL, 25.83mmol).Add azepine butane-1 then, the 4-diethyl dicarboxylate (5g, 24.6mmol) and Dimethylamino pyridine (0.305g, 2.5mmol).Solution is warming up to room temperature, and further stirred 6 hours.Finish by the definite reaction of TLC.Concentrated reaction mixture under vacuum, and add ethyl acetate precipitation di-isopropyl urea.Filtering suspension liquid.Filtrate is with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water washing.The organic layer that merges with dried over sodium sulfate and concentrate and produce crude product, and is produced the AB of 11.87g (88%) by column chromatography (50% EtOAC/ hexane) purifying.

3-[(6-amino-caproyl)-ethoxy carbonyl methyl-amino]-ethyl propionate AC

At 0 ℃ with 3-{ ethoxy carbonyl methyl-[6-(9H-fluorenes-9-ylmethoxy carbonylamino)-caproyl]-amino-(11.5g 21.3mmol) is dissolved in 20% the piperidines in dimethyl formamide ethyl propionate AB.Continue stirred solution 1 hour.Concentrated reaction mixture under vacuum, and to resistates interpolation water, and use the ethyl acetate extraction product.Crude product carries out purifying by transforming into its hydrochloride.

3-(6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-ten tetrahydrochysenes-1H-ring penta [a] phenanthrene-3-base oxygen base carbonylamino]-caproyl } ethoxy carbonyl methyl-amino)-ethyl propionate AD

With 3-[(6-amino-caproyl)-ethoxy carbonyl methyl-amino]-(4.7g 14.8mmol) is dissolved in methylene dichloride for the hydrochloride AC of ethyl propionate AC.With suspension in cooled on ice to 0 ℃.To suspension add diisopropylethylamine (3.87g, 5.2mL, 30mmol).To gained solution add the chloroformic acid cholesteryl ester (6.675g, 14.8mmol).Stirred reaction mixture spends the night.Reaction mixture is with methylene dichloride dilution, and with 10% salt acid elution.Product is by flash chromatography purifying (10.3g, 92%).

1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-ten tetrahydrochysenes-1H-ring penta [a] phenanthrene-3-base oxygen base carbonylamino]-caproyl }-4-oxo-tetramethyleneimine-3-ethyl formate AE

With potassium tert.-butoxide (1.1g, 9.8mmol) slurryization in the 30mL dry toluene.Mixture is in cooled on ice to 0 ℃, and follows to be stirred in and slowly add 5g (6.6mmol) diester AD in 20 minutes.Temperature remains on below 5 ℃ during adding.Continuation was stirred 30 minutes at 0 ℃, and added the 1mL glacial acetic acid, immediately added the NaH of 4g in 40ml water ₂PO ₄H ₂O.The gained mixture is used the 100mL dichloromethane extraction at every turn, extracts twice, and the organic extraction that merges usefulness 10mL phosphate buffered saline buffer washing at every turn, and washed twice, drying also are evaporated to dried.Resistates is dissolved in the 60mL toluene, is cooled to 0 ℃, and extract with the cold carbonate buffer solution of 3 parts of 50mL pH9.5.Aqueous extract is adjusted into pH3 with phosphoric acid, and with the chloroform extraction of 5 parts of 40mL, and merges, dry and be evaporated to dried.Resistates provides 1.9g b-ketone ester (b-ketoester) (39%) by column chromatography with 25% ethyl acetate/hexane purifying.

[6-(3-hydroxy-4-hydroxymethyl ylmethyl-tetramethyleneimine-1-yl)-6-oxo-hexyl]-carboxylamine 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-ten tetrahydrochysenes-1H-ring penta [a] phenanthrene-3-base ester AF

1 hour time durations dropwise add methyl alcohol (2mL) to the b-ketone ester AE of backflow in tetrahydrofuran (THF) (10mL) (1.5g, 2.2mmol) and sodium borohydride (0.226g, mixture 6mmol).Continuation was stirred 1 hour at reflux temperature.After being cooled to room temperature, add 1N HCl (12.5mL), (3 * 40mL) extract mixture with ethyl acetate.The ethyl acetate layer that merges crossed anhydrous sodium sulfate drying and concentrate under vacuum produce product, products therefrom is by column chromatography (10%MeOH/CHCl ₃) purifying (89%).

(6-{3-[pair-(4-methoxyl group-phenyl)-phenyl-methoxymethyl]-4-hydroxyl-tetramethyleneimine-1-yl }-6-oxo-hexyl)-carboxylamine 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-ten tetrahydrochysenes-1H-ring penta [a] phenanthrene-3-base ester AG

Glycol AF (1.25mg 1.994mmol) is by (2 * 5mL) evaporate drying in a vacuum with pyridine.Follow to stir to add anhydrous pyridine (10mL) and 4, and 4 '-dimethoxytrityl chlorine (0.724g, 2.13mmol).Being reflected at room temperature spends the night.Come termination reaction by adding methyl alcohol.Concentrated reaction mixture under vacuum, and in resistates, add methylene dichloride (50mL).Organic layer washs with the 1M sodium bicarbonate aqueous solution.With organic layer anhydrous sodium sulfate drying, filtration, also concentrated.By removing remaining pyridine with toluene evaporates.(the 2%MeOH/ chloroform is at 5%MeOH/CHCl by column chromatography for crude product ₃Middle Rf=0.5) purifying (1.75g, 95%).

Mono succinate-(4-[pair-(4-methoxyl group-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2; 3,4,7; 8,9,10; 11,12,13; 14; 15,16,17-ten tetrahydrochysenes-1H ring penta [a] phenanthrene-3-base oxygen base carbonylamino]-caproyl }-tetramethyleneimine-3-yl) ester AH

With compd A G (1.0g, 1.05mmol) with succinyl oxide (0.150g, 1.5mmol) and DMAP (0.073g, 0.6mmol) mixing, and 40 ℃ of dried overnight in a vacuum.Mixture is dissolved in the anhydrous ethylene dichloride (3mL), add triethylamine (0.318g, 0.440mL, 3.15mmol), and under argon gas stirring at room solution 16 hours.Then with it with methylene dichloride (40mL) dilution, and with ice-cold aqueous citric acid (5wt%, 30mL) and water (2 * 20mL) wash.With organic phase with anhydrous sodium sulfate drying and be concentrated into dried.Resistates so is used for next step.

Cholesterol deutero-CPG AI

With succinate AH (0.254g, 0.242mmol) be dissolved in methylene dichloride/acetonitrile (3: 2, in mixture 3mL).In this solution, be added in succession DMAP in the acetonitrile (1.25mL) (0.0296g, 0.242mmol) and acetonitrile/ethylene dichloride (3: 1,2 in 1.25mL), 2 '-dithio-two (5-nitropyridine) (0.075g, 0.242mmol).In gained solution, be added in triphenyl phosphine in the acetonitrile (0.6ml) (0.064g, 0.242mmol).The reaction mixture variable color is a bright orange.With the brief stirred solution of manual vibrator (5 minutes).Adding chain alkyl amine-CPG (LCAA-CPG) (1.5g, 61mM).Stirred suspension 2 hours.CPG is filtered by sintered funnel, and in succession with acetonitrile, methylene dichloride and ether washing.Seal unreacted amino with diacetyl oxide/pyridine.The CPG that finishes loads by adopting UV to test and measures (37mM/g).

Except the oxidation step to the cholesteryl derivative carries out with nucleic acid oligomers 5 '-end is introduced phosphorothioate bond with Beaucage reagent, the synthetic of siRNA that has 5 '-12-dodecylic acid didecyl amide group (this paper is called " 5 '-C32-") or 5 '-cholesteryl deriveding group (this paper is called " 5 '-Chol-") carries out described in WO 2004/065601.

The dsRNA expression vector

In another aspect of this invention, the E6AP specificity dsRNA molecule of regulating the E6AP activity of gene expression is expressed by the transcriptional units that inserts DNA or RNA carrier that (referring to, Couture for example, A waits the people, TIG. (1996), 12:5-10; Skillern, A. waits the people, International PCT publication number WO00/22113, Conrad, International PCT publication number WO 00/22114, and Conrad, U.S. Patent number 6,054,299).These transgenosiss can be used as linear construct, cyclic plasmid or virus vector and introduce, and it can mix host genome also as the transgenosis heredity that is integrated into host genome.Also transgenosis can be configured to and allow it as extrachromosomal plasmid heredity people such as (, Proc.Natl.Acad.Sci.USA (1995) 92:1292) Gassmann.

Each bar chain of dsRNA can be by the promoter transcription on the expression vector that separates at two, and cotransfection is gone into target cell.Alternatively, every of dsRNA other chain of branch can be by all being positioned at two promoter transcriptions on the same expression plasmid.In preferred embodiments, dsRNA is expressed as the inverted repeats that connects by the joint polynucleotide sequence, so so that dsRNA has the structure of stem and ring.

Reorganization dsRNA expression vector is generally DNA plasmid or virus vector.Express dsRNA virus vector can based on but be not limited to adeno-associated virus (referring to people such as summary Muzyczka, Curr.Topics Micro.Immunol. (1992) 158:97-129)), adenovirus is (referring to for example Berkner, Deng the people, BioTechniques (1998) 6:616), people such as Rosenfeld (1991, Cell 68:143-155)) or Alphavirus and other virus formulation known in the art Science252:431-434) and people (1992) such as Rosenfeld.Retrovirus has been used for external and/or external several genes being introduced many different cell types, comprising epithelial cell (referring to, for example Eglitis waits the people, Science (1985) 230:1395-1398; Danos and Mulligan, Proc.NatI.Acad.Sci.USA (1998) 85:6460-6464; People such as Wilson, 1988, Proc.NatI.Acad.Sci.USA 85:3014-3018; People such as Armentano, 1990, Proc.NatI.Acad.Sci.USA87:6141-6145; People such as Huber, 1991, Proc.NatI.Acad.Sci.USA88:8039-8043; People such as Ferry, 1991, Proc.NatI.Acad.Sci.USA 88:8377-8381; People such as Chowdhury, 1991, Science 254:1802-1805; People such as van Beusechem., 1992, Proc.Nad.Acad.Sci.USA 89:7640-19; People such as Kay, 1992, Human GeneTherapy 3:641-647; People such as Dai, 1992, Proc.Natl.Acad.Sci.USA89:10892-10895; People such as Hwu, 1993, J.Immunol.150:4104-4115; U.S. Patent number 4,868,116; U.S. Patent number 4,980,286; PCT applies for WO 89/07136; PCT applies for WO89/02468; PCT applies for WO 89/05345; And PCT application WO 92/07573).The recombinant retroviral vector that can transform and express the gene that inserts cellular genome can be transfected into suitable package cell line by reverse transcription virus gene group that will reorganization, and for example PA317 and Psi-CRIP produce (people such as Comette, 1991, Human Gene Therapy 2:5-10; People such as Cone, 1984, Proc.Natl.Acad.Sci.USA 81:6349).The adenovirus virus vector of reorganization can be used for infecting in the susceptible host (for example rat, hamster, dog and chimpanzee) on a large scale various kinds of cell and tissue (people such as Hsu, 1992, J.Infectious Disease, the advantage that 166:769), and also has the cell that does not need to infect mitogen activation.

Driving the dsRNA expression promoter in DNA plasmid of the present invention or virus vector can be rna plymerase i (for example ribosome-RNA(rRNA) promotor), rna plymerase ii (for example CMV early promoter or actin promoter or U1 snRNA promotor) or the common rna plymerase iii promotor (for example U6 snRNA or 7SK RNA promotor) of eucaryon; perhaps prokaryotic promoter; T7 promotor for example, condition is that expression plasmid is also encoded by the required T7 RNA polymerase of T7 promoter transcription.Promotor also can instruct transgene expression to pancreas (to regulate sequence (people such as Bucchini, 1986, Proc.Natl.Acad.Sci.USA83:2511-2515)) referring to the Regular Insulin that for example is used for pancreas.

In addition, genetically modified expression can be for example by using derivable adjusting sequence and expression system accurately to regulate, for example to some physiological regulation agent adjusting sequence of round-robin glucose level or hormone-sensitive (people such as Docherty, 1994, FASEB J.8:20-24) for example.This type of inducibility expression system that is adapted at control transgene expression in cell or the Mammals comprises by moulting hormone, by oestrogenic hormon, progesterone, tsiklomitsin, the chemical inducer of dimerisation and the adjusting of sec.-propyl-β-D1-sulfo-galactopyranoside (EPTG).Those skilled in the art can select suitable adjusting/promoter sequence based on the genetically modified desired use of dsRNA.

Generally speaking, can express as mentioned below the sending of recombinant vectors of dsRNA molecule, and remain in the target cell.Alternatively, virus vector can be used to provide the transient expression of dsRNA molecule.Examples of such carriers can be used as required repeatedly.In case express, dsRNA just is bonded to target RNA, and regulates its function or expression.Sending of dsRNA expression vector can be whole body, using for example by intravenously or intramuscular, shift out external target cell, also subsequently target cell introduced the patient again by being applied to, perhaps by other allows to introduce the method for purpose target cell arbitrarily from the patient.

The DNA plasmid of expressing dsRNA is generally with cation lipid carrier (for example Oligofectamine) or based on the carrier of non-cationic lipid (Transit-TKO for example ^TM) complex body be transfected into target cell.What the present invention had also considered to carry out dsRNA mediation during a week or longer time strikes low repeatedly lipid transfection, the different zones of the single E6AP gene of target or a plurality of E6AP genes.Successfully introducing carrier of the present invention in host cell can use multiple currently known methods to monitor.For example, transient transfection can be with reporter gene as signal, and wherein said reporter gene is fluorescent mark for example, for example green fluorescent protein (GFP).Exsomatize (ex vivo) cell stable transfection can with provide through transfectional cell to the resistance of specific environmental agents (for example microbiotic and medicine) for example the mark of hygromycin B resistance guarantee.

The special dsRNA molecule of E6AP also can insert carrier, and is used as the gene therapy vector of human patients.Gene therapy vector can pass through for example intravenous injection, topical application (referring to United States Patent (USP) 5,328,470) or by three-dimensional location (stereotactic) injection (referring to, people such as Chen for example, (1994) Proc.Natl.Acad.Sci.USA 91:3054-3057) is delivered to the experimenter.The pharmaceutical preparation of gene therapy vector can be included in the gene therapy vector in the acceptable diluent, perhaps can comprise the slow release matrix of having imbedded the gene delivery vehicle.Alternatively, when complete gene delivery vehicle can be produced by reconstitution cell fully (for example retroviral vector), pharmaceutical preparation can comprise that one or more produce the cell of genes delivery system.

Screening E6AP siRNA in the HCT-116 cell

The HCT-116 cell obtains from DSMZ (Germany microbial preservation center (Deutsche Sammlungvon Mikroorganismen und Zellkulturen)) (Braunschweig, Germany, catalog number (Cat.No.) ACC 581), and 37 ℃, contain 5%CO ₂Be incubated in the moist incubator of air and be supplemented among the McCoys (Biochrom AG, Berlin, Germany, catalog number (Cat.No.) F1015) that contains 10% foetal calf serum (FCS), 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and 2mM L-glutaminate.

For with the transfection of siRNA, with the HCT-116 cell with 2.0 * 10 ⁴The density of individual cells/well is seeded in the 96-orifice plate, and directly carries out transfection.The transfection of siRNA (30nM and 3nM are used for the single dose screening) is carried out according to manufacturers is described with lipofectamine 2000 (Invitrogen).

After the transfection 24 hours, cracking HCT-116 cell, and with Quantigene Explore test kit (Panomics, (Fremont, CA) (Genospectra in the past, Inc.)) is according to the expression level of the quantitative E6AP mRNA of standard method for Inc..E6AP mRNA level is normalized to GAP-DHmRNA.Each siRNA is collected 4 independently data points.With the irrelevant siRNA duplex of E6AP gene with comparing.The E6AP mRNA concentration that the activity of the siRNA duplex that specific E6AP is special is expressed as in handling cell is compared at the percentage ratio with the E6AP mRNA concentration in the cell of contrast siRNA duplex processing.

Hereinafter table 2 provides the result.Identified the active siRNA molecule of many target E6AP genes.

The activity of the dsRNA of table 2 target E6AP

The dsRNA of test target E6AP through chemically modified

Test identifies that through the dsRNA of chemically modified they reduce the relative capacity of the mRNA expression level of coding E6AP in cell.Adopt the above-mentioned test condition that is used for the HCT-116 cell.The E6APmRNA concentration that the activity of the siRNA duplex that specific E6AP is special is expressed as in handling cell is compared at the percentage ratio with the E6AP mRNA concentration in the cell of contrast siRNA duplex processing.

1. through the dsRNA of chemically modified

Table 3 has provided dsRNA composition of the present invention.In this table, follow hard on the same sequence that contains one or more nucleotide modifications after the sequence of unmodified.

Table 3

Capitalization: the ribonucleotide of unmodified (except T is the deoxyribonucleotide of unmodified)

Lowercase: the ribonucleotide that is partly with 2 '-O-methyl substituents at ribose

S: the position of bonding between expression thiophosphatephosphorothioate nucleosides

Chol: put together in the cholesterol moiety of 3 ' ribonucleotide.

Sense strand shown in ' duplex title ' expression is passed through and shown in the composition title that forms of the specific hybridization effect of antisense strand.

Table 4 has provided the test result of listed dsRNA in the table 3.

Table 4

The siRNA of design target HPV E1 genetic expression

Table 5 has provided dsRNA composition of the present invention.

The siRNA of test target HPV E1 genetic expression

Test unmodified and identify that through the dsRNA of chemically modified they reduce the relative capacity of the mRNA expression level of coding HPV E1 gene in cell.

The test condition that adopts is as follows: the C33A cell obtains from ATCC.The sequence clone of coding HPV16 E6 and E1 is gone into pNAS-055 carrier people such as (, Nucleic Acids Research, 31:e102,2003) Husken, be used to be expressed as YFP and merge transcript.The gained plasmid transfection is gone into the C33A cell, and select to obtain to express the stable cell lines that these merge transcript by Zeocin according to the method (Invitrogen) of manufacturers.For transfection with the siRNA of anti-HPV16 E6 or HPV16 E1, with each cell with 2.0 * 10 ⁴The density of individual cells/well is seeded in 96 orifice plates, and directly carries out transfection.The transfection of siRNA (shown in 30nM, 3nM or 300pm) is with single dose lipofectamine (Invitrogen) as described in manufacturers, carry out.

After the transfection 24 hours, lysing cell, and according to standard method Quantigene Explore test kit (Panomics, Inc. (Fremont, CA) (Genospectra in the past, Inc.)), use the expression level of the YFP mRNA that merges at the probe quantitative of YFP.The YFP mRNA level that merges is normalized to GAP-DH mRNA.To each siRNA, collect 4 independently data points.With HPV16 E1 or the incoherent siRNA duplex of E6 gene with comparing.The activity of specific siRNA duplex is expressed as the percentage ratio that fusion YFP mRNA concentration in treated cell is compared the concentration of identical transcript in the cell of handling with contrast siRNA duplex.

Table 6 shows the test result of E1 dsRNA of the present invention.

The dsRNA of design target HPV E6 genetic expression

Table 7 has provided dsRNA composition of the present invention.

The siRNA of test target HPV E6 genetic expression

Test unmodified and identify that through the dsRNA of chemically modified they reduce the relative capacity of the mRNA expression level of coding HPV E6 gene in cell.

The test condition that adopts is as follows: the C33A cell obtains from ATCC.The sequence clone of coding HPV16 E6 and E1 is gone into pNAS-055 carrier people such as (, Nucleic Acids Research, 31:e102,2003) Husken, be used to be expressed as YFP and merge transcript.The gained plasmid transfection is gone into the C33A cell, select to obtain to express the stable cell lines that these merge transcript by Zeocin according to the method (Invitrogen) of manufacturers.For transfection with the siRNA of anti-HPV16 E6 or HPV16 E1, with each cell with 2.0 * 10 ⁴The density of individual cells/well is seeded in 96 orifice plates, and directly carries out transfection.The transfection of siRNA (shown in 30nM, 3nM or 300pm) is with single dose lipofectamine

(Invitrogen) as described in manufacturers, carry out.

After the transfection 24 hours, lysing cell, and according to standard method Quantigene Explore test kit (Panomics, Inc. (Fremont, CA) (Genospectra in the past, Inc.)), use the expression level of the YFP mRNA that merges at the probe quantitative of YFP.The YFP mRNA level that merges is normalized to GAP-DH mRNA.To each siRNA, collect 4 independently data points.With HPV16 E1 or the incoherent siRNA duplex of E6 gene with comparing.The activity of specific siRNA duplex is expressed as and merges the percentage ratio that YFP mRNA concentration is compared the concentration of identical transcript in the cell of handling with contrast siRNA duplex in treated cell.

Table 8 has shown the test result of E6 dsRNA of the present invention.

Table 8

Those skilled in the art are familiar with the method and composition except that those method and compositions that the disclosure specifically provides, and this makes them put into practice the present invention in the four corner of appending claims later.

Claims

1. be used for suppressing the double stranded RNA (dsRNA) of people E6AP genetic expression at cell, wherein said dsRNA comprises at least two sequences complimentary to one another, and wherein sense strand comprises first sequence, and antisense strand comprises second sequence, described second sequence comprises the complementary region to small part mRNA that is complementary to coding E6AP substantially, and wherein said complementary region is shorter in length than 30 Nucleotide, and after the cells contacting of wherein said dsRNA and the described E6AP of expression, at least 40% suppresses described E6AP expression of gene.

2. the described dsRNA of claim 1, wherein said first sequence is selected from table 1, and described second sequence is selected from table 1.

3. the described dsRNA of claim 1, wherein said dsRNA comprises at least one modified Nucleotide.

4. the described dsRNA of claim 2, wherein said dsRNA comprises at least one modified Nucleotide.

5. claim 3 or 4 described dsRNA, wherein said modified Nucleotide is selected from: the Nucleotide that 2 '-O-methyl is modified, comprise the Nucleotide of 5 '-thiophosphoric acid ester group and be connected to the cholesteryl derivative or the terminal nucleotide of dodecylic acid didecyl amide group.

6. claim 3 or 4 described dsRNA, wherein said modified Nucleotide is selected from: Nucleotide, the morpholino Nucleotide that the Nucleotide, 2 ' of the Nucleotide that the Nucleotide, 2 ' that 2 '-deoxidation-2 '-fluorine is modified-deoxidation is modified, lock Nucleotide, dealkalize base-amido modified Nucleotide, 2 '-alkyl is modified, comprise the Nucleotide of phosphoramidate and comprise the Nucleotide of non-natural base.

7. claim 3 or 4 described dsRNA, wherein said first sequence is selected from table 1, and described second sequence is selected from table 1.

8. claim 6 or 7 described dsRNA, wherein said first sequence is selected from table 1, and described second sequence is selected from table 1.

9. the cell that comprises the described dsRNA of claim 1.

10. be used for suppressing the pharmaceutical composition of E6AP genetic expression organism, it comprises dsRNA and pharmaceutically acceptable carrier, wherein said dsRNA comprises at least two sequences complimentary to one another, and wherein sense strand comprises first sequence, and antisense strand comprises second sequence, described second sequence comprises the complementary region to small part mRNA that is complementary to coding E6AP substantially, and wherein said complementary region is shorter in length than 30 Nucleotide, and after the cells contacting of wherein said dsRNA and the described E6AP of expression, at least 20% suppresses described E6AP expression of gene.

11. the described pharmaceutical composition of claim 10, described first sequence of wherein said dsRNA is selected from table 1, and described second sequence of described dsRNA is selected from table 1.

12. the described pharmaceutical composition of claim 10, described first sequence of wherein said dsRNA is selected from table 1, and described second sequence of described dsRNA is selected from table 1.

13. be used for suppressing at cell the method for E6AP genetic expression, described method comprises:

(a) in cell, introduce double stranded RNA (dsRNA), wherein said dsRNA comprises at least two sequences complimentary to one another, and wherein sense strand comprises first sequence, and antisense strand comprises second sequence, described second sequence comprises the complementary region to small part mRNA that is complementary to coding E6AP substantially, and wherein said complementary region is shorter in length than 30 Nucleotide, and after the cells contacting of wherein said dsRNA and the described E6AP of expression, at least 40% suppresses described E6AP expression of gene; With

(b) keep the enough time of cell that step (a) produces,, thereby in cell, suppress the E6AP expression of gene with the degraded of the mRNA transcript that obtains the E6AP gene.

14. treatment, the method of the pathologic process of mediation is infected in prevention or management by HPV, described method comprises to the treatment of this kind of needs, the dsRNA of patient's administering therapeutic significant quantity of prevention or management or prevention significant quantity, wherein said dsRNA comprises at least two sequences complimentary to one another, and wherein sense strand comprises first sequence, and antisense strand comprises second sequence, described second sequence comprises the complementary region to small part mRNA that is complementary to coding E6AP substantially, and wherein said complementary region is shorter in length than 30 Nucleotide, and after the cells contacting of wherein said dsRNA and the described E6AP of expression, at least 40% suppresses described E6AP expression of gene.

15. suppress the carrier that the E6AP gene is expressed in cell, described carrier comprises the adjusting sequence of the nucleotide sequence of at least one chain that effectively is connected to coding dsRNA, the chain of wherein said dsRNA be complementary to substantially the coding E6AP to small part mRNA, and wherein said dsRNA is shorter in length than 30 base pairs, and after the cells contacting of wherein said dsRNA and the described E6AP of expression, at least 40% suppresses described E6AP expression of gene.

16. comprise the cell of the described carrier of claim 16.

17. be used for reducing the double stranded RNA (dsRNA) of people E6AP gene expression dose at cell, wherein said dsRNA comprises at least two sequences complimentary to one another, and wherein sense strand comprises first sequence, and antisense strand comprises second sequence, described second sequence comprises the complementary region to small part mRNA that is complementary to coding E6AP substantially, and after the cells contacting of wherein said dsRNA and the described E6AP of expression, reduce described E6AP expression of gene level.

18. the described dsRNA of claim 17, wherein said contact at least 40% reduces described E6AP expression of gene level.

19. the described dsRNA of claim 17, wherein said contact external with 30nM or lower carrying out.

20. be used for reducing the pharmaceutical composition of organism E6AP expression of gene level, it comprises the described dsRNA of claim 17 and pharmaceutically acceptable carrier.

21. the method for treatment HPV relative disease, described method comprises the described dsRNA of claim 17 to patient's administering therapeutic significant quantity of this kind of needs treatment.

22. the method for treatment E6AP relative disease, described method comprises the described dsRNA of claim 17 to patient's administering therapeutic significant quantity of this kind of needs treatment.

23.dsRNA it is selected from those dsRNA that table 3 is listed.

24. comprise the pharmaceutical composition of the described dsRNA of claim 23.

25. the method for treatment HPV relative disease, described method comprises the described dsRNA of claim 23 to patient's administering therapeutic significant quantity of this kind of needs treatment.

26.dsRNA it is selected from those dsRNA that table 5 is listed.

27. comprise the pharmaceutical composition of the described dsRNA of claim 26.

28. the method for treatment HPV relative disease, described method comprises the described dsRNA of claim 26 to patient's administering therapeutic significant quantity of this kind of needs treatment.

29.dsRNA it is selected from those dsRNA that table 7 is listed.

30. comprise the pharmaceutical composition of the described dsRNA of claim 29.

31. the method for treatment HPV relative disease, described method comprises the described dsRNA of claim 29 to patient's administering therapeutic significant quantity of this kind of needs treatment.

32. pharmaceutical composition, it comprises at least two kinds of dsRNA that are selected among claim 2, claim 23, claim 26 and the described dsRNA of claim 29.

33. the method for treatment HPV relative disease, described method comprises the described pharmaceutical composition of claim 32 to patient's administering therapeutic significant quantity of this kind of needs treatment.

34. the described dsRNA of claim 26 or claim 29, wherein said dsRNA comprise at least one modified Nucleotide.

35. the dsRNA of claim 34, wherein said modified Nucleotide is selected from: the Nucleotide that 2 '-O-methyl is modified, comprise the Nucleotide of 5 '-thiophosphoric acid ester group and be connected to the cholesteryl derivative or the terminal nucleotide of dodecylic acid didecyl amide group.

36. the described dsRNA of claim 34, wherein said modified Nucleotide is selected from: Nucleotide, the morpholino Nucleotide that the Nucleotide, 2 ' of the Nucleotide that the Nucleotide, 2 ' that 2 '-deoxidation-2 '-fluorine is modified-deoxidation is modified, lock Nucleotide, dealkalize base-amido modified Nucleotide, 2 '-alkyl is modified, comprise the Nucleotide of phosphoramidate and comprise the Nucleotide of non-natural base.