CN101068926A

CN101068926A - Multiple promoter expression cassettes for simultaneous delivery of RNAi agents

Info

Publication number: CN101068926A
Application number: CNA2005800139795A
Authority: CN
Inventors: P·W·罗尔温克; D·A·苏海; A·A·科雷哈洛夫
Original assignee: Benitec Ltd
Current assignee: Benitec Biopharma Pty Ltd
Priority date: 2004-03-05
Filing date: 2005-03-04
Publication date: 2007-11-07
Anticipated expiration: 2025-03-04
Also published as: ES2366126T3; CN101068926B

Abstract

The present invention provides multiple-promoter expression cassettes for simultaneous delivery of RNAi, preferably to mammalian cells in vivo.

Description

Be used for the multiple promoter expression cassettes that RNAi reagent is sent simultaneously

The mutual ginseng person of related application

It is 60/553,920 that the application has benefited from series number, and submitting day to is the U.S. Provisional Patent Application on March 17th, 2004, and series number is 60/550,504, submitting day to is the U.S. Provisional Patent Application on March 5th, 2004, and these two pieces of documents are incorporated herein by reference.

Background of invention

Use double-stranded RNA to come inhibition of gene expression to find that to medicine industry has brought change by sequence-specific mode.In Mammals, RNA disturbs, perhaps RNAi, be by 19 to 29 Nucleotide long, the double stranded rna molecule regulation and control, this double stranded rna molecule refers to the siRNA that the long dsrna molecule in the cells in vivo derives through enzymatic lysis.RNAi reagent can be in extracellular chemistry or zymetology synthetic and and then be delivered in the cell (referring to, Fire for example, etc., Nature, 391:806-11 (1998); Tuschl, etc., Genes and Dev., 13:3191-97 (1999); And Elbashir, etc., Nature, 411:494-498 (2001)); Perhaps can express in vivo by appropriate carriers in the cell (referring to, McCaffrey for example, etc. Nature Biotech.21 (6): 639-644 (2003)).

Yet the RNAi that sends unmodified in human body is to be faced with many technology barriers as therepic use effectively.At first because the nuclease in cell and the serum, the half life of injecting intravital RNA have only about 70 seconds (referring to for example, Kurreck, Eur.J.Bioch.270:1628-44 (2003)).Taked to make great efforts to increase the stability of injecting RNA by chemically modified; Yet, some have appearred because chemically changed causes the increase example of toxic effect.In a concrete example, cell can't tolerate the RNAi that per two phosphoric acid in the two strands are replaced by thiophosphatephosphorothioate dosage (Harborth, etc., Antisense Nucleic Acid Drug Rev.13 (2): 83-105 (2003)).Other obstacle comprises provides tissue specificity to send, and can send enough initiation therapeutic responses but do not have toxic RNAi reagent dosage.

For sending of RNAi had been found that many selections, comprise that use can infect target cell, and send the carrier system with expressed rna i molecule in position based on virus.Usually, the little RNA of about 70 Nucleotide is transcribed into bob folder precursor (shRNA) from the virus vector skeleton.In case transcribed, shRNA is processed into suitable viable rna i kind by the enzyme of dicing (Dicer).Attempt to develop viral target attribute to produce tissue specificity based on the route of delivery of virus,, just depend on the endogenous cell machine to produce enough RNAi kind levels and then to obtain the effective dosage of treatment in case correctly target is located.

Now, being used to of the most generally using, the virus of sending target sequence was that those develop based on retrovirus, hsv (HSV) or adenovirus (Ad) and systems of coming.All these carriers can hold sizable insertion and can be with the relevant titre production of therapeutic.Yet, all systems, all have the worry relevant with the development of cancer (Cavazzana-Calvo, etc., Science, 288:669-72 (2000)), and be attended by undesirable host immune response and in the patient, cause toxicity.Another virus that can be used for sending RNAi is adeno associated virus (AVV).

An effective application of RNAi treatment is as Anti-virus agent.Generally speaking, RNA viruses depends on the RNA polymerase that RNA/DNA relies on and duplicates.Such RNA/DNA polysaccharase is with lower rigorous degree replication-competent virus genome, and its functional consequence is to have produced the genome with unusual most sudden changes.Consequently had rapid evolution progeny virus particulate ability to escape general immunity and chemical Anti-virus agent.Therefore, similar with the effect of observed small molecules treatment, the relative potential of RNAi treatment and effectiveness may reduce owing to the virus in the long-term treatment process develops.In an example research, with the shRNA that expresses send occurred after 35 days in the body including HIV that single Nucleotide changes escape mutant (Boden, etc., J.Virol.77 (21): 11531-11535 (2003)).In another example research, in using in advance synthetic RNAi cells transfected, only just can detect poliovirus escape mutant in 54 hours after the transfection.Yet, send simultaneously two kinds at the RNAi of multiple target sequence in the virus can postpone significantly to escape mutant appearance (see Gitlin, etc., Nature.418:430-434 (2002)).

Therefore, the present technique field needs development stability, effectively RNAi treatment.The present invention has satisfied these needs in present technique field.

Summary of the invention

The present invention relates to be used for sending the composition and the method for the innovation of RNAi kind or reagent to target cell.The RNAi kind is the part of the multiple promoter expression construct preferably sent by viral delivery systems.Because three kinds or more RNAi reagent are used on each construct, the present invention can be used for using the target gene location organism with sequence difference between variant (SNPs) especially effectively, wherein the RNAi reagent of each importing can target one or more variant hypotype.Similarly, the compositions and methods of the invention also can be used for the treatment of the disease that the cause of disease by rapid sudden change causes, for example disease that is caused by the viral reagent based on RNA effectively; In other words, virus is escaped mutant and is unlikely avoided three kinds or the more effect of different RNA i sequence.

Therefore, embodiment of the present invention provide a kind of multiple promoter expression cassettes to comprise: at least three kinds of promotor/RNAi/ terminator components, wherein every kind of promotor/RNAi/ terminator component comprises promoter element, terminator element and the RNAi kind of be operably connected promoter element and terminator element, and wherein the sequence of each RNAi kind is differing from each other.Another of this embodiment preferred aspect, the sequence of each promoter element is differing from each other in multiple promoter expression cassettes.In this embodiment on the other hand, in multiple promoter expression cassettes differing from each other and/or each the terminator element of the sequence of each terminator element come from identical gene with corresponding promoter element and natural mutually in pairs.In addition, in the one side of this embodiment, the invention provides one and include the multiple promoter expression construct of the treatment carrier package being gone into the essential element of infectious viral particle.

In another embodiment of the invention, the method of one or more target set nucleic acid that a processing expresses in a cell is provided, has comprised: expressed multiple promoter RNAi expression cassette that three or more RNAi reagent suppresses one or more target set nucleic acid with one and mix virus vector and send construct so that make the RNAi of virus; Packaging virus RNAi sends construct and goes into virion; Send this virion to cell; And express from three or more RNAi reagent in this multiple promoter expression cassettes.In this embodiment of the present invention on the other hand, expressed one or more target set nucleic acid is initiation or keeps the necessary gene of morbid state, for example cancerous state in cell.In the others of this embodiment of the present invention, expressed one or more target set nucleic acid is that pathogenic agent infects cell or keeps the necessary gene of infection.Perhaps, multiple promoter RNAi expression cassette can provide by non-virus carrier, and is delivered to cell by non-viral method known in the art.

The accompanying drawing summary

For the ease of at length understanding above-described feature of the present invention, advantage and purpose, more detailed description book of the present invention, top short summary can be cited in the embodiment of accompanying drawing illustrated.Yet, should be noted that those description of drawings only are used to prove conclusively embodiment of the present invention and therefore can not be considered to limit scope of the present invention, because the present invention can comprise other same effectively embodiments.

Fig. 1 is the simplified block diagram of an embodiment that is used to send the method for RNAi kind of the present invention.

Fig. 2 A and 2B are the synoptic diagram of representing the embodiment of multiple promoter RNAi expression cassette of the present invention.

Fig. 3 A and 3B have shown that the multilist with shRNA precursor delivery RNAi reagent reaches two embodiments of carrier.Fig. 3 C has shown that comprises the embodiment that the multilist that is inserted in the filled band between promotor/RNAi/ terminator component reaches carrier.Fig. 3 D and 3E show the embodiment of the multiple promoter RNAi expression cassette of sending the RNAi that is not with the shRNA precursor.

Fig. 4 A is the concise and to the point signal of making a kind of method that is packaged into the multiple promoter RNAi expression cassette in the virion.Fig. 4 B is the concise and to the point signal of making the another kind of method that is packaged into the multiple promoter RNAi expression cassette in the virion.

Fig. 5 is the synoptic diagram of an embodiment of test recombinant chou AAV (rAAV) expression construct and luciferase report construct.

Fig. 6 is the synoptic diagram according to self complementation (scAAV) RNAi expression vector of one embodiment of the invention.

Fig. 7 is the synoptic diagram of a representative promotor test builds body and report construct.

Fig. 8 A is the genomic synoptic diagram of HCV that shows the RNAi reagent position of detecting in the experiment of the present invention's description.8B is the synoptic diagram that comprises the luciferase HCV fused replicon of the genetic elements that is used for Nonstructural Protein.8C is the synoptic diagram that comprises the luciferase HCV fused replicon of the HCV genetic elements that is used for structure and Nonstructural Protein.

Fig. 9 is the synoptic diagram that is used for two luciferase HCV fusion reporter plasmids of checking R NAi reagent.The construct of on the left comprises a 100bp HCV sequence that is fused on the luciferase genes; And comprise 3 different 100bpHCV sequences that are fused on the luciferase genes at right-hand construct.Target to one a RNAi reagent that is included in the sequence in this 100bp zone is with (if effectively) this HCV luciferase transcription product of degrading, thereby the expression of reduction (perhaps eliminating) luciferase.

Figure 10 is the graphic extension of one of embodiment of triple promoter vectors that demonstrate the unique restriction site with the module assembling that is used for multiple RNAi reagent, promoter element and terminator element.

Figure 11 A is the example of the sequence (SEQ ID NO 31) of triple promoter vectors of showing in Fig. 3 B.Figure 11 B/11C is the example of the sequence (SEQ IDNO 32) of triple promoter vectors of showing in Fig. 3 C.

Figure 12 is presented at and measures the result who is suppressed luciferase expression by the different RNAi reagent in target to five kind of different HCV100bp zone under the relative light unit (RLU).

Figure 13 has shown the result by different RNA i reagent inhibition luciferase expression who is expressed as the inhibition percent value.

Figure 14 has shown the reproducibility of check target to the test-results of four kinds of different RNA i reagent of a plurality of sections that come from the regional 100bp sequence of HCV5 '.

Figure 15 has shown the change that suppresses per-cent at target to five kinds of different RNA i reagent of a plurality of sections that come from the regional 100bp sequence of HCV5 ' luciferase expression of 24 hours and 48 hours after transfection.

Figure 16 has shown a plurality of sections five different RNA i reagent and the RNAi reagent of the sections of target 100bp sequence to HCV opening code-reading frame transfection after the luciferase expression of 44 hour and the 72 hour change that suppress per-cent of target to two different RNA i reagent of a plurality of sections of the regional 100bp sequence of HCV5 ', target to the regional 100bp sequence of HCV3 '.

Figure 17 A and 17B have shown the data of estimating three Pol III promotor intensity.A special shRNA sequence that is directed to fluorescence luciferin enzyme mRNA (McCfrey etc., 2002) is inserted under the control of above-mentioned Pol III promotor.Product plasmid DNA and luciferase reporter plasmid cotransfection are to Huh7 cell (Figure 17 A) or 293 cells (Figure 17 B).The luciferase level is measured in transfection after 72 hours.In the triple promoter constructs that come from Fig. 3 B (three constructs on each right side, map-area), demonstrate promoters driven shRNA.

Figure 18 is presented at luciferase HCV and merges the inhibition of siRNA reagent different in the reporter plasmid analysis of experiments to luciferase expression.Luciferase HCV reporter plasmid and each siRNA reagent cotransfection be to the huh7 cell, and measured luciferase activity later at 48 hours.

Figure 19 shows the activity at the selected siRNA reagent of subgene group luciferase HCV fused replicon.The pGL3 contrast DNA (being used for the contrast of transfection efficiency) of proof siRNA reagent and trace is transfection to 29 ∑ cell together.Measure the level of Lei Niliya (renilla) and fluorescence luciferin enzyme after 48 hours.

Figure 20 has shown the inhibition per-cent that comes from the luciferase signal of luciferase HCV reporter plasmid after the plasmid co-transfection with the promotor/shRNA box that comprises and have one or two active promotor.

Figure 21 has shown and has come from the inhibition per-cent that uses the luciferase signal comprise the luciferase HCV reporter plasmid that comprises C12 coding region (top), C-9 (intermediary) coding region or 5 ' 6 zones (bottom) after, the plasmid co-transfection of two or three active promotor/shRNA boxes.

Detailed Description Of The Invention

Before describing the compositions and methods of the invention, should be appreciated that the present invention is not limited to described detailed method, product and factor, because such method, instrument and preparation certainly change.Should be appreciated that also that simultaneously the term that uses only is in order to describe detailed embodiment here, and do not want to limit the scope of the invention that this scope is only limited by claims.

As what here use, " " of singulative, " one " and " this " comprise plural object, unless context is clearly stipulated in addition.Thereby for example, said " factor " is meant one or more confounding factor, and said production method comprises that those have been equivalent steps known in the field and method or the like.

Unless definition is arranged in addition, here all technology of Shi Yonging and scientific term have with the present invention under technical field in the those of ordinary skill common meaning of understanding.All publications of mentioning are incorporated herein by reference, and without any restriction, to be used for description and disclosure device, composition and method, these contents may be relevant in the described invention of the application in the publication.

In the following description, many details have been set forth to understand more completely to of the present invention.Yet the one or more those skilled in the art in these details also may realize the present invention apparently.On the other hand, the content known of those skilled in the art is not just here described.

The present invention relates to utilize single expression construct to send genetic make up thing innovation, stable and the method for at least three different RNAi reagent simultaneously to cell.This component and method provide stable, the lasting inhibition to target nucleic acid.

Usually, the present invention has used habitual molecular biology, microbiology, recombinant DNA technology, cytobiology and the virological method in this area.Such technology is illustrated in the literature fully, referring to, Maniais Fritsch ﹠amp for example; Sambrook, Molecular cloning; Experiment The chamber guide(1982); Dna clone: practicality method, volume I and 11 (D.N.Glover, ed.1985); Oligonucleotide is synthetic(M.J.Gait, ed.1984); Nucleic acid hybridization(B.D.Hames ﹠amp; S.J.Higgins, eds. (1984)); Animal cell culture(R.I.Freshney, ed.1986); With RNA viruses: practicality method, (Alan, J.Cann, Ed., Oxford University Press, 2000).

" carrier " is a replicon, and for example plasmid, phage, virus formulation body or clay wherein can assemble other DNA sections.Carrier is commonly used at transit cellization and expressible dna sections.

" promotor " or " promoter sequence " is one section can and start the DNA regulatory region transcribe in conjunction with RNA polymerase in cell, transcribe to as if the small nut of polynucleotide or polypeptid coding sequence such as messenger RNA(mRNA), nuclear candy body RNAs, nucleolar RNA s or by the RNA of rna plymerase i, II or any any kind of of transcribing of III.

When such nucleic acid during transfered cell this cell for example, have the complex body of transfection reagent or be packaged in the virion just by nucleic acid external source or allogenic or plasmid institute " conversion ", " transduction " or " transfection " as one.The DNA of this conversion may or can not integrate (covalently bound) to the genome of cell.For eukaryotic cell, in the cell of stable conversion, transfering DNA has been integrated in the host cell karyomit(e) or has maintained outside the karyomit(e), so that this transfering DNA is inherited by daughter cell during cellular replication or this transfering DNA is that the non-cell that duplicates, breaks up wherein exists persistent episome.

Term " RNA interference " or " RNAi " generally are meant such process, and wherein double stranded rna molecule or short hairpin RNA have changed the expression of nucleotide sequence, and they have basic or homology completely.Term " RNA kind " or " RNAi reagent " are meant the RNA sequence of the initiation RNAi of one section uniqueness; And term " RNAi expression vector " is meant the carrier that comprises three above RNAi kinds according to embodiments of the present invention.

Fig. 1 is a simplified flow chart, and it has shown the step of method 100, wherein can use according to multiple promoter RNAi expression construct of the present invention.At first, in step 200, constructed the multiple promoter RNAi expression cassette of a target to the specified disease target.Secondly, in step 300, multiple promoter RNAi expression cassette is connected to suitable virus and sends in the construct.This viral RNA i expresses and to send construct and be packaged in the virion then in step 400, and this virion is delivered in the target cell that needs treatment in step 500.Details that these steps comprise and composition describe in detail below.

Multiple promoter RNAi expression construct based on virus according to the present invention can utilize many working specifications well known in the art to produce by synthetic or zymetology means, and utilize the standard recombinant dna technology to come purifying, these technology are documented in, for example, people such as sambrook The molecule gram Grand: lab guideSecond edition, cold spring port are published, the cold spring port, NY (1989) and according in for example U.S. HHS portion, national health research centre (NIH) about the recombinant DNA research described rules of guide and carry out.In a preferred embodiment, this multiple promoter RNAi expression cassette is to utilize phosphoramidite or analogue according to working specification synthetic well known in the art.

Fig. 2 A and 2B are the sketches of multiple promoter RNAi expression cassette according to embodiments of the present invention.Fig. 2 A has shown to have three promotors/embodiment of the multiple promoter expression cassettes (10) of RNAi/ terminator component (being presented at 20), and Fig. 2 B shows an embodiment of the multiple promoter expression cassettes (10) with five promotors/RNAi/ terminator component (being presented at 20).P1, P2, P3, P4 and P5 represent promoter element.RNAi1, RNAi2, RNAi3, RNAi4 and RNAi5 represent five different RNAi kind sequences.T1, T2, T3, T4 and T5 represent termination element.Multiple promoter RNAi expression cassette according to the present invention can comprise three or more promotor/RNAi/ terminator component, wherein the number of the promotor that comprises at any multiple promoter RNAi expression cassette/RNAi/ terminator component is subject to, for example, selected delivery system (for example, some viruses, such as AAV, relatively strict size restriction is arranged) the packing size; Cytotoxicity, and maximal effective dose (for example, the validity when the expression of four RNAi sequences is treated to be used as is identical with the effect of ten RNAi sequences expression).

Three or more RNAi kinds all has different sequences in the box that includes promotor/RNAi/ terminator component; That is to say that RNAi1 RNAi2, RNAi3, RNAi4 and RNAi5 have nothing in common with each other each other.Yet the promoter element in any box may be the same (that is to say that for example, the sequence of two or more P1, P2, P3, P4 and P5 may be the same); All promotors of box inside can have nothing in common with each other each other arbitrarily; Perhaps have the promoter element that only occurs in arbitrary box inside once and occur twice or the set of above promoter element.Similarly, the terminator element in arbitrary box may be the same (that is to say that for example, two or more sequences may be the same among T1, T2, T3, T4 and the T5, such as 4 or the successive stretching area of more T residues); All terminator elements of arbitrary box inside can differ from one another; Perhaps have the terminator element that only occurs in arbitrary box inside once and occur twice or the set of above terminator element.Preferably, all different with the terminator element at each promoter element that comprises in the promotor/RNAi/ terminator component of any box to reduce the possibilities that the DNA recombination event takes place between component and/or box.In addition, in a preferred embodiment, the promoter element and the terminator element that are used for each promotor/RNAi/ terminator component are complementary each other; That is to say that promotor and terminator are taken from them and are present in wherein same gene natively.

Fig. 3 A, 3B and 3C show multiple promoter RNAi expression construct carrier, wherein include the selectivity embodiment of the multiple promoter RNAi expression cassette that can express short shRNAs.ShRNAs is that short double chain form wherein has justice to be connected by hairpin loop with antisense strand.In case express, shRNAs is formed to the RNAi kind.Picture frame A, B represent three different promoter elements with C, and arrow has been indicated the direction of transcribing.TERM 1, TERM2 represent three different terminator sequences with TERM3, and shRNA-1, shRNA-2 represent three different shRNA kinds with shRNA-3.Multiple promoter RNAi expression cassette in this embodiment reaches the arrow that is labeled as TERM3 from the picture frame that is labeled as A.In three promotor/RNAi terminator components (20) of Fig. 3 A demonstration each all is on the same direction in this box, Fig. 3 B demonstration simultaneously is for shRNA-1 and the shRNA-2 promotor/RNAi/ terminator component in one direction, shRNA-3 promotor/RNAi/ terminator component (for example, is transcribed on two chains that occur in carrier) in the opposite direction.

Fig. 3 C shows by the DNA region separation to increase each box of the distance between promotor/RNAi terminator component.The DNA of this insertion is called as " filling " DNA, can be the length between 5-5000 Nucleotide.Between promotor, one or more stuffer can be arranged.For multiple stuffer, they can be same or different length.This fills the preferably different sequence of dna fragmentation.This filling dna fragmentation can be used to increase the size of multiple promoter box of the present invention so that allow it to be suitable for becoming corresponding delivery vector.The length of this stuffer is that the size by the specific support relevant with the multiple promoter carrier needs defined.For example, stuffer amounts to long 4000 Nucleotide (nt) so that satisfy the big or small needs of AAV carrier rightly in one embodiment.In other embodiments, stuffer amounts to 2000nt so that satisfy the big or small needs of self complementary AAV carrier rightly.Other variant also can use.

Fig. 3 D and 3E have shown multiple promoter RNAi expression construct, and it includes the selectivity embodiment of the multiple promoter RNAi expression cassette that can express the RNAi kind that does not have hairpin loop.In two width of cloth figure, P1, P2, P3, P4, P5 and P6 represent promoter element (arrow has been indicated transcriptional orientation); And T1, T2, T3, T4, T5 and T6 represent termination element.In two width of cloth figure, RNAi1 sense strand and RNAi1 antisense strand (a/s) are complementary equally, and RNAi2 sense strand and RNAi2 antisense strand (a/s) are complementary, and RNAi3 sense strand and RNAi3 antisense strand (a/s) are complementary.

In Fig. 3 D embodiments shown, it is to transcribe (by P1, P2 and P3) from a chain that all three RNAi have adopted sequence, and these three RNAia/s sequences are transcribed (by P4, P5, P6) from complementary strand simultaneously.In this specific embodiment, the termination element of RNAi1 a/s (T4) drops on promotor P1 and RNAi 1 has between the adopted sequence; And the termination element of RNAi1 (T1) drops between RNAi 1 a/s sequence and its promotor P4.These motifs be multiple so that if the top chain that shows among Fig. 3 D is called (+) chain and the bottom is called (-) chain, this moves element from left to right and will meet with P1 (+), T4 (-), RNAi1 (justice and a/s are arranged), T1 (+), P4 (-), P2 (+), T5 (-), RNAi2 (justice and a/s are arranged), T2 (+), P5 (-), P3 (+), T6 (-), RNAi3 (justice and a/s are arranged), T3 (+) and P6 (-) successively.

In another shown embodiment of Fig. 3 E, all RNAi have justice and antisense sequences to transcribe from same chain.Fig. 3 A may be used to application-specific to the embodiment of shown any one multiple promoter RNAi expression cassette of 3E to those skilled in the art, and can be group thing or variant.

In some embodiments, can the variable promotor of working strength.For example, the use of three or more strong promoter (such as Pol III type promotor) may cause burden by pair cell, by for example exhausting the component of transcribing necessary available Nucleotide or other cell.In addition or in addition, the use of several strong promoters can cause the toxic level that RNAi reagent is expressed in cell.Thereby the one or more promotors in multiple promoter RNAi expression cassette may be weaker than other promotor in the box in some embodiments, and perhaps all promotors can be lower than expressed rna i reagent on the level of maximum rate in this box.Promotor also can by or do not modified by molecular engineering, perhaps opposite, for example, obtain transcribing of more weak level by controlling element.

Promotor can be tissue-specific or cell-specific.The promotor that term " tissue-specific " is meant when being applied to promotor can instruct the target nucleotide sequence to carry out the selective expression to specific type of tissue (for example liver), and lacks the expression of same target nucleotide sequence on the other hand in another kind of specific type of tissue (for example brain).Such tissue-specific promoter comprises such as Ick, myogenin or thyl.The promotor that term " cell-specific " is meant when being applied to promotor can instruct the target nucleotides sequence to be listed in selective expression in the cell of particular type, and in same in-house another kind of specific cell type, lack on the other hand same target nucleotide sequence expression (referring to, for example, Higashibata, Deng. J.Bone Miner.Res.Jan19 (1): 78-88 (2004); Hoggatt, etc., Circ.Res., Dec.91 (12): 1151-59 (2002); Sohal, etc., Circ.Res.Jul89 (1): 20-25 (2001); And Zhang, etc., Genome Res.Jan 14 (1): 79-89 (2004)).Term " cell-specific " refers to also when being used for promotor that promotor can promote the target nucleotides sequence to be listed in the selective expression in the zone of single organization inside.Additionally, promotor be composing type with regulatable.In addition, promotor can be modified so that have different specificitys.

This promotor (for example, heat-shocked, chemistry, light or the like) under the condition that lack to stimulate that term " composing type " is meant when being used for promotor still can instruct transcribing of nucleotide sequence that one section operability connects.Usually, constitutive promoter can instruct the expression of the encoding sequence of arbitrary cell and tissue basically.The promotor that is used for transcribe rna i kind is constitutive promoter preferably, such as promotor at ubiquitin, CMV, β Actin muscle, histone H 4, EF-lalfa or pgk gene, it is controlled by rna plymerase ii, perhaps the promoter element of being controlled by rna plymerase i.In preferred embodiments, use promoter element by rna plymerase iii control, such as U6 promotor (U6-1, U6-8, U6-9 etc.), H1 promotor, 7SL promotor, human Y promotor (hY1, hY3, hY4 (and referring to Maraia, etc., Nucleic Acids Res22 (15): 3045-52 (1994)) and hY5 ((referring to Maraia, etc., Nucleic Acids Res3552-59 (1994)), the functional hybridization and the combination of human MRP-7-2 promotor, adenovirus VA1 promotor, human tRNA promotor, 5s ribosome-RNA(rRNA) promotor and above-mentioned any promotor 24 (18):.

Additionally in some embodiments, perhaps preferred those allow the promotor of RNAi kind inducible expression.This area has been known many systems that are used for inducible expression and has been used such promotors, yet comprises and be not limited to system that tsiklomitsin replys and lac operon-repressor system (referring to WO 03/022052 A1; With US 2002/0162126 A1), moulting hormone regulation system, the perhaps promotor of regulating by the regulator or the metallothioneins promotor (regulating) of glucocorticosteroid, progesterone, oestrogenic hormon, RU-486, steroidal, Triiodothyronine, cyclic amp, cytokine, vitamin d family by inorganic metal.

One or more enhanser also may be present in this virus multiple promoter RNAi expression construct to increase the expression of target gene.Be suitable for the enhanser that embodiment of the present invention is used comprise those Apo E HCR enhansers of having described recently, cmv enhancer (referring to, Xia etc., Nucleic Acids Res31-17 (2003)), reach other enhanser well known in the art.

In one embodiment of the invention, the ApoE enhancer element can be added on the multiple promoter box of the present invention.The ApoE enhanser is the enhancer element that derives from apo E or ApoE of about 155 base pairs (bp).The ApoE enhanser of one or more copy can be added in multiple promoter box of the present invention first, upstream or downstream (upstream or the downstream that perhaps surpass three promotors) of second and/or the 3rd promotor.ApoE is a lipophorin, can regulate and control combination, internalization and the catabolism of lipoprotein particle and is at low-density lipoprotein (ApoB/E) acceptor with at the aglucon of hepatic tissue ApoE acceptor.With the hereditary enhanser of ApoE gene-correlation be the controlling elements of eucaryon, can increase transcribing of liver specificity nucleic acid.The ApoE enhanser may be positioned at the position of liver specificity promotor upstream or downstream 2000 Nucleotide, and may have one or more copy.

By the RNAi sequence of multiple promoter RNAi expression cassette of the present invention coding cause little interferential RNA s (that is to say mammalian cell do not have toxic weak point, double-stranded RNA s) expression.As long as they do not have showed cell toxicity, the length of RNAi kind of the present invention does not have specific limited.RNAi can be, for example, 15 to 49bp length, and preferred 15 to 35bp length, and preferred 19 to 29bp length.The double-stranded RNA part of RNAis is homology completely, perhaps owing to sequence mispairing (is not complementary at the corresponding nucleotide on each chain), protrusion (lacking corresponding complementary nucleotide on the chain) or the like cause comprising the non-matching part.Such non-matching part does not have to form or bring into play under the inconsistent prerequisite of effect with RNAi significantly and can tolerate at them.

As long as RNAi can reticent effectively target gene, can be flush end or cohesive end (cantilevered) according to the terminal point of RNAi kind of the present invention.The end structure of cohesive end (cantilevered) not merely is limited to 3 ' cantilever, but as long as product RNAi can induce the RNAi effect, 5 ' cantilevered structure is includable.In addition, cantilevered Nucleotide number can be that any amount is as long as product RNAi can induce the RNAi effect.For example, if any, cantilever may be made up of 1 to 8 Nucleotide; Preferably it is made up of 2 to 4 Nucleotide.

The present invention use the RNAi kind can have stem-ring structure precursor (shRNA) wherein the end of double-stranded RNA connected by strand joint RNA.The length of the single-stranded loop part of shRNA can be 5 to 20bp, and preferably 5 arrives 9bp.

Any one nucleotide sequence of transcribing can be the target at multiple promoter RNAi expression cassette of the present invention.Possible target at this RNAi is such gene, yet for example be not limited to developmental character gene (for example, adhesion molecule, cyclin kinase inhibitor, Wnt family member, Pax family member, wing spiral family member, cytokine/lymphokine and their acceptor, growth/differentiation factor and their acceptor, neurohumor and their acceptor); Oncogene (for example ABL1, BCL1 BCL2, BCL6, CBFA2, CBL, CSF1 R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES); Tumor suppressor gene (for example APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1); And enzyme (for example acc synthase and oxydase, ACP desaturase and hydroxylase, the ADP-glucose pyrophosphorylase, ATPases, ethanol dehydrogenase, amylase, amyloglucosidase, catalase, cellulase, chalcone synthase, chitinase, cyclo-oxygenase, decarboxylase, dextrinase, DNA and RNA polymerase, tilactase, dextranase, glucose oxidase, particle is in conjunction with starch synthase, GTPase, helicase, hemicellulase, intergrase, inulinase, saccharase, isomerase, kinases, Sumylact L, lipase, lipoxygenase, N,O-Diacetylmuramidase, the nopaline synthase, the octopine synthase, Rohapect MPE, peroxidase, Phosphoric acid esterase, Phospholipid hydrolase, Starch phosphorylase, phytase, the plant-growth regulator synthase, polygalacturonase, proteolytic enzyme and peptase, Starch debranching enzyme, recombinase, reversed transcriptive enzyme, RUBISCO, topoisomerase, and zytase); Structure gene such as the housing and the envelope protein matter of virus; The gene of bacterium relates to such as those and duplicating or constitutional features, perhaps duplicates or the gene of constitutional features from other relating to of pathogenic agent; In addition, multiple promoter RNAi expression cassette of the present invention can be used for target to specific sequence, described specific sequence is to cause symptom allelotrope in the disease (such as SCA) of euchromosome domination, the allelotrope that causes the Huntington pathology perhaps causes the glue protogene allelotrope of osteogenesis imperfecta peculiar.An important aspect of the present invention be by the virus infection that siRNA removes can not cause to cells infected any injury (Gitlin, etc. Nature418:430-434 (2002)).This feature of the present invention is different from method of the prior art, wherein remove come from the destruction that causes infected cell under the apoptotic effect that mammalian hosts virus can cause in immunity system or by virus (Guidotti etc. Annu.Rev.Immunol.19:65-91 (2001)).Thereby this aspect of the present invention provides an effective RNAi reagent to remove virus under acellular causes a disease condition.

Genetic sequence with target target nucleotide sequence is the sequence that the RNAi kind is selected on the basis; And be benchmark preferably with target nucleotide sequence conservative region.For example, select at the treatment virus infection or when making up the RNAi sequence of RNAi vaccine, preferred sequence is those sequences conservative between this virus kind even between the subspecies.Virus variation is very fast as known in the art, and the selection of conserved sequence will keep the effect of RNAi as far as possible in for some time.When selecting the RNAi sequence with treatment cancer or other disease, preferred sequence is those sequences conservative between gene or oncogene variant.

The method that is used for sequence homology comparison and RNAi sequence selection has been known in this area.Identity per-cent determines and can finish by mathematical algorithm between two or more sequences.Preferably, nonrestrictive example is the algorithm (1988) of Myers and Miller; The search similarity method (1988) of Pearson and Lipman; And the algorithm of Karlin and Altschul (1993).Preferably, use a computer and carry out these mathematical algorithms.Such example includes, but are not limited to: the CLUSTAL gene program on the Personal Computer (can be from Intelligenetics, Mountain View obtains among the Calif .); ALIGN program (version 2 .0), GAP, BESTFIT, FASTA, Megalign, (use Jotun Hein, Martinez, the Needleman-Wunsch algorithm), TFASTA in DNAStar Lasergene (see www.dnastar.com) and the Wisconsin genetics software package, version 8 (can be from GeneticsComputer Group (gcg), 575 Science Drive Madison Wis. obtain among the USA).Use the homology comparison of this program can use the parameter of default parameter or operator's selection to carry out.The CLUSTAL program was described in detail by Higgins.The ALIGN program is a benchmark with mayer and Miller's; And blast program is a benchmark with the algorithm of Karlin and Altschul; The software of carrying out the BLAST analysis can obtain (http://www.ncbi.nim.nih.gov/) by the National Center forBiotechnology Information.

In order to carry out sequence alignment, a sequence is as consensus sequence, and sequence to be tested is compared with it.When using a sequence alignment algorithm, to be tested and consensus sequence input computer is specified the subsequence coordinate if necessary, and specified sequence algorithm routine parameter.Then, be benchmark with specified program parameter, sequence comparison algorithm calculates the sequence identity percentage of sequence to be tested (s) with respect to consensus sequence.

Usually the target sequence that is suppressed by RNAi need be between target sequence and RNAi molecule sense strand very high sequence homology.In some embodiments, it is about 70% that such homology is higher than, and can be to be higher than about 75%.Preferably, it is about 80% that homology is higher than, and be higher than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In the embodiment of using multiple promoter RNAi expression construct target virus infection, 15 to 30 successive homology of nucleotide sequence may not have arrival to surpass 90% even 80% level between the subspecies genome that virus is different--even in conservative zone--.Target sequence and the sequence homology between the RNAi sense strand at some subspecies can be 80% or lower in this case.

On the other hand, when the target gene of biology does not demonstrate kind, during height sequence homology between subspecies or the variant, multiple promoter RNAi expression construct of the present invention is useful especially, and each the RNAi kind in the multiple promoter RNAi expression cassette all can be used for locating the different sites of target gene (s) or variant or subspecies set at this moment.

Except the conservative region with target sequence was the RNAi sequence selected of benchmark, selecting the RNAi sequence can other the factor be benchmark.Although there have been many effort to design at differentiating sequence--the feature that this discriminating sequence is based on target sequence is (for example, GC percentage composition, translation initiation codon position or based on the sequence similarity of the sequence library of the RNAi homology that is used for searching for suggestion) and be effective at RNAi--choice criteria, can't foretell that the countless possible RNAi candidate sequences corresponding to target can cause actual RNA silent reaction with very high confidence level at present.Opposite, usually, produce and detect special candidate rna i polynucleotide sequence to determine whether the interference that target is expressed is initiated out.

A main problem of current antiviral therapy is the resistance variant to occur, usually be called escape mutant (Gitlin etc. J.of Virol.79; 1027-1035, (2005)).An aspect of of the present present invention can be suppressed the escape mutant of appearance.The selection of many RNAi sequence to the treatment virus infection is based on from the escape mutant in the cell that is infected by strand RNAi sequence in some embodiments of the present invention.The escape mutant that occurs is to be determined by the treatment of using the expression construct that comprises the RNAi single stranded sequence to carry out after cell is by virus infection.The cell that includes resistance virus is collected and viral genome is checked order.The main mutant that sequencing result shows is used to resist virus and suppresses.Produce multiple promoter RNAi expression construct of the present invention, it includes based on the RNAi sequence of target gene order and the additional point mutation sequence that is used to resist the RNAi treatment.

As described, the exercisable terminator element that is connected to of the RNAi coding region of multiple promoter RNAi expression cassette.In one embodiment, terminator comprises the extension region with four or more thymidine residues.In a preferred embodiment, the terminator element of all uses all is different, and is complementary with promoter element, and both come from same gene.Such terminator comprises the terminator of SV40 poly A, Ad VA1 gene, 5S ribosomal RNA gene and human t-RNA.In addition, promotor and terminator can mix and mate, as common use RNA pol 11 promotors and terminator.

In addition, can dispose multiple promoter RNAi expression cassette and make multiple clone site and/or specific limited site to locate, can remove easily or replace such as promotor, RNAi and terminator element by certain strategy.And multiple promoter RNAi expression cassette can be by small oligonucleotide component assembling, and this component is used has localized tactfully restriction site and/or complementation to glue end.One of carrier is carrier has comprised the plasmid with multiple connexon according to embodiments of the present invention, and wherein all sites all are specific (though this are not absolute requirement).

Then, each promotor all is inserted into and has formed a basic box with three or more promotors in the specific site that designs, and all promotors can have different directions.Then, annealing primer has formed threefold representation vector construction body on the specific site that is inserted into each promotor downstream.Such insertion can move to, and for example, use is on the AAV skeleton of two specific restriction enzyme sites (identical or different) of threefold representation carrier insertion site flank.

In the step 300 of Fig. 1, multiple promoter RNAi expression cassette is connected on the delivery vector.That multiple promoter RNAi expression cassette is inserted and be used for various kinds of cell transduce effectively and the construct of expressed rna i reagent be derive from virus and can hold virus and send.Can the be well known in the art any genetic engineering technique of the production of this construct is realized, includes but not limited to, standard round pcr, oligonucleotide are synthetic, digestion with restriction enzyme, connection, conversion, plasmid purification and dna sequencing.Construct preferably includes, and for example, multiple promoter RNAi expression construct is packaged to the required sequence of virion and/or allows multiple promoter RNAi expression construct to be inserted into the genomic sequence of target cell.The virus formulation body also can comprise the gene that allows virus replication and propagation, although so in preferred embodiments gene is a cis.In addition, the virus formulation body can comprise gene or the genetic sequence in the natural or modified genome in any known life entity.For example, preferred virus formulation body has comprised and has been used for the sequence of duplicating on bacterium.

This construct also can comprise additional genetic elements.Can be included in component kind in the construct is not subjected to any restriction and can be selected by those skilled in the art.For example, additional genetic elements can comprise reporter gene, such as one or more fluorescent mark albumen (as GFP or RFP), enzyme that is easy to detect such as beta-galactosidase enzymes, luciferase, β-glucuronidase, E.C. 2.3.1.28 or excretory embryo alkaline phosphatase; The albumen such as hormone or the cytokine that perhaps can be used for immunoassay.Other genetic elements that can be used for embodiment of the present invention comprises the element of those coded proteins--described protein can bring the selective growth advantage to cell, for example adenosine deaminase, aminoglycoside phosphotransferase, Tetrahydrofolate dehydrogenase, Totomycin-β-phosphotransferase perhaps encode and can be provided at the protein of the biosynthesis ability that lacks in the auxotroph.If comprise the reporter gene along multiple promoter RNAi expression cassette, so inherent ribosome entry site(RES) (IRES) sequence also can be included in wherein.Preferably, be connected to additional genetic elements operability and controlled by promotor/enhanser independently.

Viral delivery systems based on suitable virus can be used to send multiple promoter RNAi expression construct of the present invention.In addition, can use the hybrid virus system.The selection of viral delivery systems depends on multiple parameter, the target tissue of for example sending, the transduction efficiency of system, pathogenic, immunity and toxicity or the like.Consider the diversity that can use multiple promoter RNAi expression cassette of the present invention institute interferential target disease, the single viral system of none can be fit to all application.When selecting viral delivery systems to be applied among the present invention, it is very important selecting such system, the virion that comprises multiple promoter RNAi expression cassette in the described system preferably: 1) renewable and stable propagation; 2) can be purified to very high titre; With 3) can regulate and control targeted delivery (multiple promoter RNAi expression construct is delivered to destination organization or organ and do not have large-scale diffusion); 4) can express in the mode of composing type or adjustable type.

Generally speaking, be incorporated in the host cell chromosome (oncogenic retrovirus and beans shape virus) or be present in the nucleus mainly as the outer episome of karyomit(e) according to genome, five kinds of modal viral delivery systems of gene therapy that are applicable to are divided into two classes.This differentiation is every kind of important factor at the suitability of specific end use carrier of decision; Nonconformity type carrier can, in some cases, the genetic expression that in non-proliferative cell, regulate to continue, if but in the splitted cell, need to keep stable hereditary change, integrating vector just becomes the instrument of selection.

For example, in one embodiment of the invention, used virus from Parvoviridae family.Parvoviridae is one and has the genomic strand of about 5000 length of nucleotides, non-coating dna virus family.One of member is adeno associated virus (AAV), this virus be the dependency parvovirus need and other virus (general is adenovirus or simplexvirus) coinfection so that initiation and keep an infectious cycle that productive efficiency is arranged.Lack under the situation of a such helper virus, still the have the ability combination regulated through acceptor and internalization of AAV infected or transduceed to target cell, infiltrate non-division and division stage cell nucleus.

In case enter nucleus, virus opens shell and different this transforming gene of formal representation--wherein the most stable is the annular monomer from many.AAV will be integrated in the cellular genome of stable transduction of 1-5% (Nakai, etc., J.Virol.76:11343-349 (2002).The expression of this transforming gene can be stable especially and one about using AAV to send in the research of factors IX, the dog phantom type factor IX proteins of continuous expression treatment level still after this virus is directly invaded 5 years.Because progeny virus is not infected by AAV and produces under the situation that lacks helper virus, the degree of transduction only is subject to infects this viral initiator cell.This feature makes AAV become preferred gene therapy vector of the present invention.And, be different from retrovirus, adenovirus and hsv, AAV demonstrate shortage to the mankind's pathogenic and toxicity (Kay, etc., Nature.424:251 (2003) and Thomas, etc., Nature Reviews, Genetics4:346-58 (2003)).

Usually, the genome of AAV comprises only two genes.At least four different protein that " rep " genes encoding uses in dna replication dna." cap " gene product is comprised three protein of this capsid with generation by montage differently.When genome was packaged to the virus at initial stage, only inverted terminal repeat sequence (ITRs) was essential sequence; Rep and cap can delete and can be replaced by the heterologous sequence of candidate from this genome.Yet this protein need duplicate and will be packaged to the virion at initial stage based on the allos construct of AAV in order to make, and rep and cap protein must be cis.Provide this subsidiary function by helper virus coinfection in general, also show as cis with the form of one or more DNA expression plasmid such as above-mentioned adenovirus or simplexvirus.Two genes since this genome is usually only encoded, not surprisingly, as means of delivery, AAV is limited in the bale capacity of 4.5kb strand.Yet though this big or small restriction may limit those genes that are used for gene therapy that can transmit, it does not have influence packing and the expression of short sequence such as RNAi on the contrary.

The purposes of AAV in RNAi uses show in such experiment, wherein AAV be used for external transmission shRNA with the expression that suppresses p53 and Caspase 8 (Tomar etc., Oncogene.22:5712-15 (2003)).Along with cloning suitable sequence in digestible AAV-2 carrier, in the HEK293 cell, produced communicable AAV virion and be used to infect HeLa S3 cell.Living Caspase 8 and p53 level have a dose-dependent minimizing in having shown.People such as Boden also use AAV external transmission shRNA be suppressed at HIV in the tissue culture system duplicate (Boden, etc., J.Virol.77 (21): 115231-35 (2003)), as what estimate by the p24 product that in the substratum of remainder, exists.

Yet, the also mandatory declaration of technology barrier when using AAV to be used for multiple promoter RNAi expression construct as carrier.For example, different percentage ratio human population can have the neutralizing antibody at certain AAV serotype.Yet, since several AAV serotypes are arranged, some type being carried the rapid minimizing of individual per-cent of neutralizing antibody, other serotype can be used or intend type and also can use.Have at least eight kinds of different serotypes to be identified, though and other many types are separated is not also described fully.Another restriction is, as one possible at the immunoreactive result of AAV, only can carry out once based on the treatment of AAV; Yet, use serotype that replace, the non-human origin can allow repeat administration.The genomic composition of route of administration, serotype and transmission all can influence tissue specificity.

AAV system another restriction in use that utilizes the unmodified of multiple promoter RNAi expression construct is but that the transduction energy efficiency is low.Stable transduction may be limited to the cell of 5-10% in the body.Yet this area has known that diverse ways promotes stable transduction level.A method is to utilize the plan type, wherein uses the cap protein pack AAV-2 genome that derives from other serotype.For example, replace AAV-5 cap gene by using the AAV-2 counterpart, people such as Mingozzi in about 15% liver cell, increased stable transduction (Mingozzi, etc., J.Virol.76 (20): 10497-502 (2002)).People such as Thomas use AAV8 housing gene transduceed in vivo 30% mouse liver cell (Thomas, etc., J.Virol.In press).People such as virionGrimm ( Blood.2003-02-0495) use AAV-1, AAV-3B, AAV-4, AAV-5 and AAV-6 limit the plan type of AAV-2 to be used for Study on tissue culture.The highest transforming gene expression level is by the plan C-type virus C particle inductive that uses AAV-6; Produced than 2000% the transforming gene expression nearly of AAV-2 height.Thereby the present invention plans to use the AAV of a plan typeization to realize transduction level highly, corresponding increasing that brings RNAi multiple promoter expression construct to express.

Another viral delivery systems that uses promotor RNAi expression construct of the present invention is a viral system based on Retroviridae (Retroviridae) family.Retrovirus comprises the single stranded RNA animal virus with two specific characteristics.At first, retroviral genome is a diploid, is made up of the RNA of two copies.The second, the enzyme reverse transcription of this RNA process virion association is to double-stranded DNA.Therefore this double-stranded DNA or provirus can be integrated into host genome and be delivered to progeny cell as the stable integrated element of this host genome from parent cell.

In some embodiments, slow virus is to be used for retroviral preferred member of the present invention.Usually use blister talcum viral glycoprotein (VSV-G) to intend the type lentiviral vectors, and risen and be derived from: Human Immunodeficiency Virus (HIV)--the cause of disease of people's AIDS-like disease (AIDS); Sheep encephalitis-chronic progressive external pneumonia, it can cause encephalitis (visna) or pneumonia in sheep; Equine infectious anemia virus (EIAV), it causes autoimmune hemolytic anemia and encephalopathic in horses; Feline immunodeficiency virus (FIV), it causes immune deficiency in feline; In ox, cause lymphadenopathy and lymphocytotic bovine immunodeficiency virus (BIV); The immunodeficiency virus of ape (SIV), it causes immune deficiency and encephalopathic in the non-human Primates.Carrier based on hiv virus usually keeps＜5% parent's genome, and＜25% genome is to merge to enter the packing construct, has minimized the generation possibility of recovering replication HIV.Development by self-inactivation carrier has further increased biological safety, self-inactivation carrier has comprised in the deletion of the controlling element in long terminal repeat downstream, has eliminated carrier and has mobilized transcribing of the packaging signal that needs.

The reverse transcription of retrovirus rna gene group occurs in the tenuigenin.Be different from the slow virus cDNA--that C-type retrovirus and other virokine be combined with each other and be called as preinitiation complex--can cross that displacement takes place nuclear membrane and Unseparated Cell is gone in transduction.As if the constitutional features of this virus cDNA--DNA aileron--help to enter efficiently nucleus.This aileron depends on central polypurine zone (cPPT) complete on the pol gene that is positioned at virus, so the carrier of most slow virus origin has kept this sequence.Slow virus has tropism widely, low struvite potential, and cause forming integrative vector.Main restriction is to integrate to cause tumour to generate in some utilizations.Using the main advantage transgenosis in most tissue or cell type of lentiviral vectors is to continue.

Being used for the construct based on slow virus of expressed rna i reagent preferably includes sequence from long terminal repetition (LTRs) 5 ' and 3 ' of slow virus.More preferably this virus formulation body includes an inactivation coming from slow virus or 3 ' LTR of self-inactivation.LTR can make by self-inactivation according to any means well known in the art.In a preferred embodiment, the U3 element of 3 ' LTR comprises the sequence of enhanser disappearance, and preferred enhanser is TATAbox, Sp1 and NF-kappa B site.As the result of 3 ' LTR oneself inactivation, the provirus that is incorporated into host genome will comprise 5 ' LTR of an inactivation.The LTR sequence can be the LTR sequence from any slow virus kind.In addition, can use promoter sequence in the virus formulation body to replace U3 sequence from slow virus 5 ' LTR.This can increase the titre of the virus of recovering from package cell line.Also can comprise simultaneously an enhancer sequence.

Well known to those skilled in the art other virus or non-viral system can be used to transmit multiple promoter RNAi expression vector of the present invention to target cell, yet comprise and be not limited to stably keep the genetically deficient adenovirus-transposon carrier of encoding viral transforming gene in vivo (referring to Yant by being integrated into host cell, Deng. Nature Biotech.20:999-1004 (2002)); Derive from sindbis alphavirus (Sindbis virus) or Xi Menli the gram forest virus (Semliki forest virus) system (referring to Perri, etc., J.Virol.74 (20): 9802-07 (2002)); Derive from the system of Avian pneumo-encephalitis virus or Sendai virus; Perhaps lack the dna sequence dna of bacterium little cyclic DNA carrier (referring to Chen, etc., Molecular Therapy.8 (3): 495-500 (2003)).The little cyclic DNA of describing in the open No.2004/0214329 of the U.S. has been showed the carrier that is used for transcribing Nucleotide with continuing the height level.The characteristics of this circular vectors are to lack the bacterium sequence of expression silencing, and may comprise a unidirectional locus specificity recombinant product sequence except that expression cassette.

In addition, hybrid virus system can be used to make up two or more viral system's useful property.For example, the site-specific integration machine of wild-type AAV can join the efficient internalization of adenovirus and examine the target characteristic by coupling.The AAV that exists in adenovirus or the simplexvirus has experienced the replication cycle of a high yield; Yet, lacking under the situation of subsidiary function the specificity site of AAV genome conformity to the karyomit(e) 19.The genomic integration of AAV needs AAV rep protein expression.Because conventional AAV carrier has been deleted all virogenes that comprise rep, they can not be integrated on the karyomit(e) 19 specifically.Yet this feature can develop in suitable crossing system.In addition, non-viral genetic elements can be used for being implemented in the required characteristic requirement of delivery system of a virus, allows the genetic elements of locus specificity reorganization such as those.

In the step 400 of Fig. 1, multiple promoter RNAi expression construct is packaged in the virion.Any means well known in the art can be used to produce the copy that its genome of infectious virus particle includes this virus multiple promoter RNAi expression construct.Fig. 4 A and 4B demonstration are used for packing the other method of multiple promoter RNAi expression construct of the present invention to the virion of being sent.Method in Fig. 4 A has been used packing cell, this cell is stably expressed according to cis viral multiple promoter RNAi expression construct is bonded to the needed virus protein of virion, and be used for specific virus delivery system necessity or preferably other sequence (for example, duplicate the sequence that needs, structural protein and virus assembling) and that enter at tissue or viral origin or artificial source's part.In Fig. 4 A, multiple promoter RNAi expression cassette is connected to (step 300) on the viral delivery vector, and product virus multiple promoter RNAi expression construct is used for cell (step 410) in the transfection packing.Packing cell is the replication-competent virus sequence then, expresses virus protein and packaging virus multiple promoter RNAi expression construct to infectious virus particle (step 420).Package cell line can be the arbitrary cell system that can express virus protein, however comprise be not limited to 293, HeLa, A549, PerC6, D17, MDCK, BHK, bing cherry, phoenix, Cf2Th or other those skilled in the art's clone of knowing or making arbitrarily.For example, in U.S. Patent No. 6,218, the package cell line of describing in 181.

Additionally, can not stably express the clone of necessary virus protein can be by two or more construct cotransfections to realize effective production of functional particle.Such construct includes viral multiple promoter RNAi expression construct, and another plasmid (s) includes the nucleic acid of the coding permission functional virus of cells produce (duplicating and pack construct) institute proteins necessary and other subsidiary function.The method that Fig. 4 B shows has been used and has been used to pack virus replication product and the packaging gene that those are not stably expressed.In this example, promotor RNAi expression construct is connected in the viral delivery vector (step 300) then the carrier that duplicates required virus sequence and infectious virus particle product with one or more expression and carries out cotransfection (step 430).The cellular replication virus sequence is expressed virus protein and packaging virus multiple promoter RNAi expression construct (step 420) to infectious virus particle.

Package cell line or duplicate and pack construct and may not express the env gene product.In these embodiments, the gene of encoded packets membranin can provide on an independent construct and that is to say and viral multiple promoter RNAi expression construct cotransfection.Because envelope protein is responsible for the host range of virion to a certain extent, virus can be the plan type.As what describe in the above, " plan typeization " virus is to have the virion that comes from virus envelope protein, and this virus is not the virus of this genome origin.Those skilled in the art can select the delivery system that suitable plan type is used to use and the cell of target.Except that giving the specificity host range, the plan type of selection can be permitted virus and is concentrated into very high titre.Additionally virus can be had a liking for environment envelope protein matter and carries out plan type (for example, have a liking for the environment coating and only allow to infect the murine cell, and the ambitendency coating allow to infect human and the murine cell) to the specificity kind is infectious by being used for restriction.In addition, the part of genetic modification can be used for the cell-specific target, such as being used for hepatocellular asialoglycoprotein, perhaps is used for receptor modulators bonded siderophilin).

After producing in package cell line, the virion that comprises multiple promoter RNAi expression cassette carries out purifying and quantitative (titration).The purifying strategy comprises density gradient centrifugation, and is perhaps preferred, column chromatography.

Viral multiple promoter RNAi expression cassette of the present invention can be especially suitable for use as handles treatment of diseases means or prophylactic vaccine.For example, multiple promoter RNAi expression construct can be introduced in cancer cell or the tumour to suppress to keep carcinogenic disease/necessary expression of gene of tumour generation phenotype.Similarly, multiple promoter RNAi expression construct can be introduced in the cell of infecing pathogenic agent such as virus to suppress one or more necessary expression of gene of pathogenic agent of keeping.Multiple promoter RNAi expression construct can be to come the target initiation or keep disease or the required gene of symptom as vaccine.

Viral multiple promoter RNAi expression construct of the present invention can be used for the treatment of cancer, comprise solid tumour and leukemia, comprise: apudoma, choristoma, branchioma, malignant carcinoid syndrome, the innocent tumour heart trouble, malignant tumour (for example, Walker, the basilar cell, the basophilia squamous cell, brown Pierre Si, conduit, the Ehrlich tumour, original position, Krebs 2, the Merkel cell, cement, non-small cell lung, oat cell, nipple, inocarcinoma, segmental bronchus, bronchogenic spread, squamous cell, and transitional cell), the histocyte disorder, leukemia (B cell for example, cell mixing, null cell, the T cell, chronic T cell, the HTLV-II association, acute lymphoblastic, chronic lymphocytic, mastocyte and spinal cord), pernicious hystiocytosis, Hodgkin's disease, little immunoproliferating, non-Hodgkin lymphoma, plasmoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofibroma, adenolymphoma, sarcocarcinoma, chordoma, cranium pharynx knurl, dysgerminoma, progonoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, gland cancer, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, lymphangiosarcoma, myosarcoma, myxosarcoma, malignant tumor of ovary, rhabdosarcoma, sarcoma (for example, You Yin, experiment, Kao Boxi, and mastocyte), neoplasm (for example, bone, breast, Digestive tract, colorectum, liver, pancreas, hypophysis, testis, eye socket, head, and neck, central nervous system, the sense of hearing, pelvis, respiratory tract and apparatus urogenitalis) neurofibroma, and cervical atypism hyperplasia, and at other situation treatment wherein cell do not become dead or transform.In addition, multiple promoter RNAi expression construct of the present invention can be used to the associating of therapeutic modality with other, such as chemotherapy, surgical operation, psychrotherapy, high temperature, radiotherapy or the like.

Participate in that pathogenic agent is duplicated, pathogenic agent is sent or keep infectious gene and can carry out target through viral multiple promoter RNAi expression construct.Viral multiple promoter RNAi construct like this can be used for the treatment of and is in by cell in the infectious danger of pathogenic agent (for example vaccine) or infected cells.Can comprise virus by the pathogenic agent of multiple promoter RNAi expression construct of the present invention and method treatment from Parvoviridae family, papovaviridae (comprising papillomavirus or the like), Adenoviridae, herpetoviridae (comprising herpes virus type 1 to 7), Poxviridae, have a liking for Hepadnaviridae, small nut grain nucleic acid virus (Ke Saji A and Ke Saji B virus and ECHO virus), calicivirus section, Reoviridae, the toga Viraceae, (encephalitis), flavine Viraceae (encephalitis), Arenaviridae, Retroviridae, bunyaviridae, coronaviridae, orthomyxovirus section, Paramyxoviridae, Rhabdoviridae and inovirus section, general bacterium, mycobacterium, fungi, malaria, trypanosome Schistosoma or the like.

In the step 500 of Fig. 1, multiple promoter RNAi expression construct is delivered to needs the treatment express cell.Multiple promoter RNAi expression construct of the present invention can then be put into animal to influence therapeutic process then at external introducing cell, perhaps directly is administered to organism, organ or cell by vivo medicine-feeding.By sending of virus infection is preferred delivering method; Yet the arbitrarily suitable delivering method that can be used for multiple promoter RNAi expression construct all is spendable.The carrier that utilizes any suitable working specification to include the multiple promoter box can be administered to mammalian hosts, and many such working specifications have been known in this area.

Nucleic acid can be introduced in tissue or the host cell by many approach, comprises that virus infection, microinjection or vesica merge.Injection also may be used for the muscle administration, as Furth etc. Anal.Biochem.115 (205): 365-368 (1992) describes.Nucleic acid can cover on the gold grain, and by the partickle bombardment device described in the document or " particle gun " (referring to falling into Tang etc., Nature.356:152-154 (1992)) be delivered to intradermal, wherein gold grain is bombarded to skin cells then by the DNA packaging.

Another can be used for delivering method of the present invention comprised use as Daviset etc. in U.S. Patent No. 6,509, the Cyclosert of description in 323 ^TMTechnology.Cyclosert ^TMTechnology platform is based on the glucose cup-shaped cycle repetition molecule that is called as cyclodextrin.Cyclodextrin molecular " cup " can form " inclusion mixture " with other molecule, make to Cyclosert ^TMPolymer and other parts make up to improve stability or to increase the target part becomes possibility.In addition, usually cyclodextrin to be considered to the mankind are safe (independent cyclodextrin improve the solubleness of FDA approval and IV medicine at large)

The carrier that comprises the multiple promoter box can be prepared into by dissolving in water-based or non-aqueous solution, suspension and emulsification and be used to inject or the goods of administration, and non-aqueous solution can be for example oil, synthetic glycerin fatty acid ester, high-grade aliphatic ester or propylene glycol; And if desired, can use conventional additive for example expanding material, isotonic agent, suspension agent, emulsifying agent, stablizer and sanitas.

In addition, the carrier that comprises the multiple promoter box can be prepared into pharmaceutical composition together with suitable, pharmaceutically acceptable carrier or diluent.In the pharmacopedics formulation, the carrier that comprises the multiple promoter box can individually dosed or and other pharmacy activity component associating or combination medicine-feeding.Those skilled in the art can be readily appreciated that the dosage level of the carrier that comprises the multiple promoter box can be used as the function of expression level of RNAi kind in the relative difficulty or ease of the natural character of delivery system, target cell transformation, the target cell or the like and changes.

Embodiment

Hepatitis C virus (HCV) is a kind of disease that can use multiple promoter RNAi expression construct treatment of the present invention.--nearly 400 ten thousand--infected HCV now according to the statistical information of disease control and prevention center compilation, almost 2% American.Originally, most HCV people infects and do not demonstrate any symptom; Yet, also finally cause liver cirrhosis or hepatocellular carcinoma above 80% the hepatic diseases chronic and progressively development that will develop into.At U.S. HCV is to cause the main reason of liver transplantation and cause 8000 to 10000 American death every year.World Health Organization's estimation has the infected above 100,000,000 seven thousand ten thousand in the whole world, at the 10-30% of some national infection rate up to total population.

HCV is just strand coating RNA viruses, belongs to flavine Viraceae family.Virion entered cell after the infectious cycle of HCV usually started from receptor-mediated combination and internalization.In tenuigenin, open after the shell, comprise genomic RNA positive-sense strand can with host cell machine translator direct interaction.Lack methylating of 5 ' cap, RNA has formed a sufficient secondary structure at 5 ' non-translational region (UTR), and wherein the UTR direct combination that inherent ribosome entry site(RES) (IRES) is provided and allowed the 40S subunit is with the initial step as translation program.

The HCV genome, the length of about 9600 Nucleotide, a long opening code-reading frame of the polyprotein by name of having encoded (in Fig. 8 A, showing).Virus protein is produced with the precursor forms that connects from polyprotein, and polyprotein is cut into matured product by the enzyme in multiple virus and the cell subsequently.The structural protein of having encoded in these genes comprise core and envelope glycoprotein, and name is because they are complete structural constituent in the progeny virus particle like this.Non-institutional protein provides for example RNA polymerase of dependenc RNA of indispensable function, also is produced.The virion copy machine is set up in the tenuigenin of infected cell and just rna transcription can be become the minus strand intermediate.Therefore, HCV genome cell is simultaneously as the template of self-replication and be used to translate the proteic messenger RNA(mRNA) of encoding viral.Minus strand is transcribed back positive chain RNA, has therefore increased the quantity of normal chain copy in the cell.In this stage, positive chain RNA can interact again with the host cell machine translator or, if the accumulation of enough structural proteins has been arranged, be packaged into virion.Along with the release from cell, virus repeats in whole infectious cycle.

Embodiment 1: be used for sending in the body exploitation of the AAV-2 expression vector of shRNA sequence

The shRNA via infectious particle is sent test before, suitable expression plasmid is fabricated and verifies.Have at least two features to need to consider when design multiple promoter RNAi expression construct: 1) construct must be packed effectively and enter the progeny virus particle; With 2) plasmid must provide high-caliber shRNA to express.In addition, in order to detect multiple promoter RNAi expression construct, the means of assessment transfection and transduction efficiency must be arranged.

The AAV-2 carrier that is mixed (gut) by rep and cap sequence provides the skeleton that is used for viral RNA i expression construct (referring to the rAAV carrier hereinafter).This carrier has been widely used in the AVV research and the needs that high-level efficiency is packed have had good understanding.U6 and H1 promotor have been used for the expression of shRNA sequence, though there is report to show that same shRNA at each promotor independent drive has inhibition very in various degree.Yet, if such variation exists the promotor in the vector construction to replace at an easy rate.

In fact the same with any viral delivery systems, the rAAV carrier must satisfy the certain criteria size so that packed effectively.Generally speaking, the rAAV carrier lengths must be 4300-4900 Nucleotide (McCarty, etc. Gene Ther.8:1248-1254 (2001)).When the rAAV carrier is lower than this restriction, necessary adding stuffer (Muzyczka, etc. Curr.Top.Microbiol.Immunol.158:970129 (1992)).Additionally, rAAV multiple promoter RNAi expression construct must load two or more multiple promoter RNAi expression cassettes.

In the embodiment of described here rAAV carrier, about 400 Nucleotide of each promotor/RNAi/ terminator length component have stayed enough spaces and have been used for holding a plurality of promotors/RNAi/ terminator assembly at each expression cassette.Additionally, can in rAAV multiple promoter RNAi expression construct, add the transfection efficiency of one or more selected marker boxes, and allow to use the rAAV expression construct of sending to come the transduction efficiency of target cell is quantized through the virion that infects for use in assessment rAAV expression construct.

Initial check expression vector has driven the expression of shRNA kind, this shRNA kind according to proved have inhibition from the sequence of the luciferase activity ability of report construct design (referring to, Elbashir, etc. Embo.J.20 (23): 6877-6888 (2001)).The element of RNAi box comprises promotor, shRNA and terminator sequence, is short and utilizes long, complementary oligonucleotide independently from new assembling, and this oligonucleotide uses multiple clone site to be cloned in the virus vector then.The commercial expression vector codes luciferase function product that can get as the report thing so that proof shRNA regulates and control the target sequence ability of (as shown in Figure 5) downwards.

Though the shRNA at luciferase before was verified, at first assess external by the effect of the shRNA that rAAV sent.Utilize standard technique will detect and report the cell of construct transfection to permission.The rAAV expression construct of using incoherent shRNA sequence to replace luciferase specificity shRNA is used as in test negative contrast.By the level of fluorescence microscopy assessment selected marker thing and the relative percentage of direct estimation transfection efficiency thus.In order to assess the inhibition activity of shRNA, use the activity of the commercial reagents box measurement luciferase of standard.Additionally, use quantitative PCR in real time analysis (Q-PCR) technical Analysis from the parallel laboratory test plate, to gather in the crops and purified RNA.With respect to the activity that records from the cell pyrolysis liquid that uses uncorrelated shRNA kind to handle, the activity minimizing above 90% has shown that shRNA has the height function.

Carry out subsequent experimental with the influence of assessment shRNA for the luciferase reporting system, this system is transfected to mouse liver, exists with people such as McCaffrey Nature.The work of delivering on the 418:38-39 (2002) is similar.The nucleic acid that is delivered to mouse by the water power infection protocol mainly is positioned at liver.Very similar with the principle of domination cotransfection in cell cultures, when coming from the many plasmid of mixture injection usually all expression construct penetrate into identical cell.Therefore, though proved the tail vein injection program can only the transfection liver in 5-40% liver cell (McCaffrey, etc. Nature Biotech.21 (6): 639-644 (2003)), cotransfection also allows reporting system and expression construct are delivered in the identical cell.

Carry at injecting altogether with the rAAV expression construct of the shRNA sequence of luciferase and the report construct of coding luciferase genes.In the animal of having accepted negative control, carry expression construct and the injection altogether together of report construct of uncorrelated shRNA.Measure the activity of luciferase based on the lysate that results from a liver part.Q-PCR measures and histologic analysis obtains standardized data with the expression of determining labelled protein thereby the remainder of liver is used for.Other method of assessment transfection efficiency comprises that ELISA that the serum from mouse is carried out measures, wherein use the 3rd be used for secretory protein such as human α 1-synalbumin (hAAT) (Yant, etc. Nature Genetics.25:35-41 (2000), also referring to McCaffrey, etc. Nature Biotech.21 (6): marker plasmid cotransfection mouse 639-644 (2003)).

In case confirmed that expression construct has function equally in the mouse model of cell in vitro culture systems and use naked DNA plasmid co-transfection, become infectious viral particle from rAAV expression construct packing and begin to test.The AAV that can get from commerce does not have assistant subsystem and makes infectious particle, and this no assistant subsystem needs three independent expression construct carry out cotransfection, comprises 1) express rAAV construct at the shRNA (flank is AAV ITRs) of luciferase; 2) construct of coding AAV rep and cap gene; With 3) include the expression construct of producing the required auxiliary adenoviral gene of infectious titer.After the standard purification program, virion can be used for experiment.

Before mouse is injected into the rAAV particle, set up a cover reporting system in the mouse liver.Hydrodynamic transfection is used to send luciferase report construct and for the expression plasmid at hAAT of transfection efficiency difference between the control animal.Mouse is allowed to recover several days so that produce the report thing activity of enough levels.

In liver, proved conclusively after the luciferase report activity, the rAAV particle has been injected normal C57B1/6 mouse by portal vein or tail vein.The rAAV particle that carries uncorrelated shRNA expression construct is used as negative control.Originally, use higher dosage (2 * 10 ¹²Vector gene group (vg)) injects mouse, in experiment subsequently, reduce dosage to produce dose-response curve.After seven to ten days, put to death mouse, collect the sample of liver and serum.From isolating liver, utilize luciferase analysis and previously described QPCR program to decide the related levels of liver luciferase activity and RNA.Additionally, assess the efficient of transduction by the labelled protein in the measurement liver organization serial section.

Result of experiment has very wide scope.The verified hydrodynamic transfection program in front may cause the hepatocellular transfection of 5-40%.Use the AAV-2 delivery program hepatocellular result that transduces to show the transduction efficiency of 5-10%.Though AAV may preferentially transduce by the same liver cell storehouse of initial tail intravenous injection program transfection, the cell subclass of each technique influence may be nonoverlapping.If the generation former instance can be seen with respect to the minimizing that a luciferase activity is arranged for the mouse of uncorrelated shRNA kind transduction.If the latter's situation takes place, can not observe the reduction of luciferase activity so.

Must prove the AAV particle sent by the threefold representation construct in vitro inhibition luciferase--HCV fusion rotein report thing.The report construct institute transfection that the tissue culture cells of permission is described in detail by Fig. 9.In addition, each cotransfection mixture all has the plasmid of coding hAAT.After cultivating 48 hours, the infectious particle pair cell that use carries at triple promotor shRNA expression plasmids of HCV carries out administration.The AAV particle that comprises triple promoter constructs of expressing three kinds of uncorrelated shRNA kinds is used as negative control.The activity of measuring luciferase highly has function with the shRNA that checking AAV sends.

Embodiment 2: strengthen the modification of AAV transduction liver organization efficient

Though the verified carrier based on AAV can be sent the sequence that needs to liver cell, the level relatively that generally speaking occurs in the transduction in those tissues is suitable low.Current use AAV-2 does not obtain relevant result to send and to express in the hemophilia clinical study of blood factor IX.In order to treat hemophilia, The key factor only is to replenish secretory protein to make it reach the level that can bring into play curative effect.Replenishing like this may occur in minority and can express in the transducer cell of a large amount of desirable proteins.Yet because the RNAi mechanism of action is intracellular and its effect can not directly be propagated at iuntercellular, the efficient that must increase transduction is so that AAV can express shRNA and be used for the treatment of.

People such as McCarty can produce self complementary AAV carrier (scAAV), this carrier in its capsid, have simultaneously the normal chain of same expression cassette and minus strand ( Gene Ther.8:1248-1254 (2001)).This is by suddenling change 5 ' ITR and keep that 3 ' ITR is constant to be finished.By sudden change or deletion is terminal separates other non-basic AAV sequence of site, thereby eliminated possible reorganization between wild-type AAV and this construct, having created a dna profiling, wherein to be replicated in 3 ' ITR initial.In case copy machine arrives 5 ' ITR, can not take place to separate and duplicate to last till 3 ' ITR.Result product has normal chain and complementary minus strand, still can pack effectively.Use the scAAV carrier, the liver cell of transduction be increased to total hepatocellular 30% (Fu, etc. Molec Therapy.8 (6): 911-7. (2003)).When between the endoplasmic reticulum vacuole, sending, transduceed by the scAAV particle above 50% cerebellum pears shape neurone.People such as Thomas report self complementary carrier can in mouse liver, produce the luciferase transgene expression level higher 50 times than the corresponding strand AAV expression vector that injects mouse liver with same dose (Thomas, etc., J.Virol.(inpress)).Though descend slightly, the relative different degree of expressing between carrier in nearly a year after the injection maintains 20 times.

Used similar strategy herein.The amount of space of--comparing suitable little with other viral delivery systems--owing to, can be used for the delivery of therapeutic sequence with the related size restriction of packing rAAV multiple promoter RNAi expression construct since use scAAV reduce by half.Therefore, Da Xiao restriction is lowered to 2250 Nucleotide rather than can packs 4500 Nucleotide.No matter these, the size that multiple promoter RNAi expresses the h box allows such construct.Fig. 6 has shown the diagram of the inner main element of ScAAV.

Other of AAV delivery system modified the efficient also be used to greatly increase transduction, comprises that use is from the Cap albumen packing rAAV-2 vector gene group of other serotype virion with production plan typeization.Even the advantage that obtains through using plan type strategy is arranged, may only be increased to 15% of total population to the limit of liver cell transduction efficiency.Yet 12 other AAV serotype is separated and differentiate, but does not also have can specifically describe to appreciable degree.For example, one of them be AAV-8, it is separated from the rhesus monkey heart tissue at first.Done the time spent to what transduction had measuring new cap albumen, the virus of plan typeization is created out, and wherein strand AAV-2 genome is with AAV-8cap plan type.Carrier carries the LacZ gene so that the relative efficiency of behind the infectious particles of assessment injection increase dosage mouse liver being transduceed.To result's summary (Thomas, etc. J Virol.78 (6): 3110-22. (2004)) be presented in the following table 1:

The dose response of table 1 AAV-2/2 and AAV-2/8 (%beta-gal male liver cell)

Carrier	Dosage (v.g./mouse)
	Dosage (v.g./mouse)					5×10 ¹⁰	3×10 ¹⁰	1.8×10 ¹¹	3.9×10 ¹²	7.2×10 ¹²
	AAV-2/2 LacZ	0.6±0.4％	3.0±0.5％	8.1±1.0％	8.9±1.0％	5×10 ¹⁰	3×10 ¹⁰	1.8×10 ¹¹	3.9×10 ¹²	7.2×10 ¹²	NA
AAV-2/8 LacZ	AAV-2/2 LacZ	0.6±0.4％	3.0±0.5％	8.1±1.0％	8.9±1.0％	8.1±1.8％	14.9±3.4％	65.8±9.1％	NA	97.4±0.3％	NA

When the dosage of the contrast AAV-2/2 particle that injects increases, hepatocellular transduction there is the increase of an appropriateness.Yet the upper limit of transduction stably remains near 10%.Surprisingly, the AAV-2/8 particle of plan typeization 8% the liver cell of under the situation of minimum dosage, having transduceed; Compare with AAV-2/2 contrast dosage low 30-80 doubly.Additionally, surpassed transduction efficiency in the increase that depends on dosage, and efficient exceeds 97% when maximum dose level at AAV-2/2 at the transduction efficiency of AAV-2/8.Transduction efficiency in this scope can be delivered to RNAi the cell of organization internal effectively.

RAAV RNAi expression construct has been made in the similar modification of AAV.After mixing these simple modifications, produce a large amount of virions and be used for check in mouse model system.Check at following rAAV RNAi experiment virus: strand AAV-2/2; Strand AAV-2/8 is from complementary AAV-2/2; With from complementary AAV-2/8.

With to carry the corresponding virion of rAAV carrier of expressing uncorrelated shRNA sequence manufactured and be used for negative control.The luciferase activity huge minimizing of level and the increase of transduction efficiency relatively is associated.

Embodiment 3A: select and check the RNAi reagent that is directed to luciferase HCV fusion plasmid

Selecting shRNAs is not a simple proposition as the useful treatment means at HCV.Except producing the problem of escaping mutant, high mutation frequency has caused the quite large-scale sequence polymorphism in carrying virulent infected person crowd, has produced to have the nearly different genotype of 31-34% in nucleotide sequence.Based on full-length gene group sequence alignment, hypotype (kind in a given genotype scope) has the difference of 20-23%.Therefore, the viral genome zone with high conservative preferably determine and be selected for and guarantee wide range of therapeutic suitability.As an aligned sequences and select the example of treatment relevant range how, from public's database, obtain corresponding to 30 full length sequences of HCV genotype 1b virus and use Jotun Hein method and MegAlign analysis software (DNASTAR) is compared.Zone with high conservative is identified out, for example the zone (Nucleotide 75-112) in 5 ' UTR.

In order to select candidate sequence, carried out one to all disclosed independent total lengths or near the comparison of the HCV sequence of total length; Current have 200 such sequences of surpassing representing all known genotype.Current exist severally be used for selecting and develop the candidate region of RNAi treatment and fully confirmed 5 ' and 3 ' UTR zone belong to conservative region at the HCV genome.Although known because cell translation conjugated protein or these non-coding sequences of modulin potential spatial obstacle may not be ideal target sequences, the RNAi target of a height functionalization that people such as Yokota are verified to 5 ' UTR of replicon system ( EMBO Rep.4 (6): 602-608 (2003)).Though confirm that several zones that have absolute certitude within one 21 nucleosides (the corresponding size of the target sequence in the shRNA kind) stretching area are useful, the analysis of data shown that the conservative of a kind of like this degree is can not occur in a plurality of hypotypes of idiotype even related to all genotype.Therefore, selection can comprise having like this and surpasses 80% zone and kept fragment in the absolute conservative genome.In the final construct of candidate, the expression of three independent shRNAs has compensated the sequence mutability before clinical, has allowed to be included in the combination therapy in the single delivery vector.

Additionally, if those conservative regions that meet choice criteria are not differentiated in to the genotypic analysis of all HCV, sequential analysis can be confined to genotype (1a and 1b), they account for the U.S. and infect 3/4ths of population nearly except that Africa, are topmost genotype in worldwide.In addition, up-to-date, effective anti-HCV treatment adds the use of uniting of the Interferon, rabbit of polyoxyethylene glycol and virazole (a guanosine-analogue), is quite low at the efficient of genotype 1, but very high at another genotype efficient.Thereby, in patient's population of volume, exist replacing the huge needs of therapy.Because comparison only shows homology,, when the final RNAi reagent that selection is used to check, be used so other choice criteria is intersected specific shortage such as relative GC content with when the search sequence database.

For example, in an experiment, to comparing from the multiple sequence of HCV hypotype 1a and 1b.Several conservative regions are confirmed to be has enough length so that therefrom select RNAi reagent to be used for check (greater than 19 nucleosides).5 ' UTR and 3 ' UTR zone are the most conservative zones.Since the homologous zone has been identified and sufficiently long, between different genotype, also compare.Can select generally conservative zone in conjunction with two comparisons.Some zones, for example the long stretching area of A ' s or U ' s, G ' s and C ' s is not considered, because they are unsuitable for the target by RNAi reagent place, the zone of remaining " qualified " is used for further selection.(opening code-reading frame) has only a general conservative zone to be identified to can be used for the HCV genotype that all are considered in whole coding region; So it is conservative in hypotype 1a and 1b that the sequence of selecting in most of examples is those.

In case the zone of " qualified " is identified, according to RNAi reagent antisense strand 5 ' end should have than 3 ' terminal low this standard of free energy selects one RNAi sequence." adjacency pair free energy " rule is used to calculate in 5 of all potential RNAi reagent of having selected ' and 3 ' free energy of terminal 5 terminal nucleosides so far.As a result of, confirmed 30 potential RNAi reagent altogether: 10 5 ' UTR (5 '-n), 12 at opening code-reading frame ORF (C-n), and 83 ' UTR (3 '-n) (see Table 2).The relative position of these RNAi target site is presented among Fig. 8 A in the HCV genome.

Table 2:RNAi sequence

RNAi reagent	The RNAi sequence ^#	SEQ ID NO.	The Luc-HCV reporter plasmid	The HCV position
RNAi reagent	The RNAi sequence ^#	SEQ ID NO.	The Luc-HCV reporter plasmid	The HCV position	5′-1	gCTGTGAGGAACTACTGTCT	SEQ ID NO.1	20	IRES 43-62
5′-2	GTCTAGCCATGGCGTTAGT	SEQ ID NO.2	-	IRES 77-95	5′-1	gCTGTGAGGAACTACTGTCT	SEQ ID NO.1	20	IRES 43-62
5′-2	GTCTAGCCATGGCGTTAGT	SEQ ID NO.2	-	IRES 77-95	5′-3	GGAGAGCCATAGTGGTCTG	SEQ ID NO.3	16，20	IRES 131-149
5′-4	GCGGAACCGGTGAGTACAC	SEQ ID NO.4	16	IRES 150-168	5′-3	GGAGAGCCATAGTGGTCTG	SEQ ID NO.3	16，20	IRES 131-149
5′-4	GCGGAACCGGTGAGTACAC	SEQ ID NO.4	16	IRES 150-168	5′-5	GTCTGCGGAACCGGTGAGTA	SEQ ID NO.5	16	IRES 146-165
5′-6	GCGAAAGGCCTTGTGGTACT	SEQ ID NO.6	16，17	IRES 270-289	5′-5	GTCTGCGGAACCGGTGAGTA	SEQ ID NO.5	16	IRES 146-165
5′-6	GCGAAAGGCCTTGTGGTACT	SEQ ID NO.6	16，17	IRES 270-289	5′-7	GATAGGGTGCTTGCGAGTG	SEQ ID NO.7	16	IRES 295-313
5′-8	GAGGTCTCGTAGACCGTGCA	SEQ ID NO.8	16，17	IRES 319-338	5′-7	GATAGGGTGCTTGCGAGTG	SEQ ID NO.7	16	IRES 295-313
5′-8	GAGGTCTCGTAGACCGTGCA	SEQ ID NO.8	16，17	IRES 319-338	5′-9	gCTTGTGGTACTGCCTGATA	SEQ ID NO.9	-	IRES 279-298
5′-10	gCTGCCTGATAGGGTGCTTG	SEQ ID NO.10	17	IRES 289-307	5′-9	gCTTGTGGTACTGCCTGATA	SEQ ID NO.9	-	IRES 279-298
5′-10	gCTGCCTGATAGGGTGCTTG	SEQ ID NO.10	17	IRES 289-307	C-1	AGATCGTTGGTGGAGTTTA	SEQ ID NO.11	-	Core 427-445
C-2	gTTGGGTAAGGTCATCGATA	SEQ ID NO.12	-	Core 696-714	C-1	AGATCGTTGGTGGAGTTTA	SEQ ID NO.11	-	Core 427-445
C-2	gTTGGGTAAGGTCATCGATA	SEQ ID NO.12	-	Core 696-714	C-3	GCCGACCTCATGGGGTACAT	SEQ ID NO.13	18	Core 732-752
C-4	GGTTGCTCTTTCTCTATCT	SEQ ID NO.14	-	Core 852-870	C-3	GCCGACCTCATGGGGTACAT	SEQ ID NO.13	18	Core 732-752
C-4	GGTTGCTCTTTCTCTATCT	SEQ ID NO.14	-	Core 852-870	C-5	GGGATATGATGATGAACTG	SEQ ID NO.15	-	NS1 1300-1318
C-6	GGATGAACCGGCTAATAGC	SEQ ID NO.16	-	NS4B 6085-6113	C-5	GGGATATGATGATGAACTG	SEQ ID NO.15	-	NS1 1300-1318
C-6	GGATGAACCGGCTAATAGC	SEQ ID NO.16	-	NS4B 6085-6113	C-7	GGAGATGGGCGGCAACATC	SEQ ID NO.17	-	NS5A 7046-7064
C-8	GTCTTCACGGAGGCTATGA	SEQ ID NO.18	-	NS5B 8610-8629	C-7	GGAGATGGGCGGCAACATC	SEQ ID NO.17	-	NS5A 7046-7064
C-8	GTCTTCACGGAGGCTATGA	SEQ ID NO.18	-	NS5B 8610-8629	C-9	GTCAACTCCTGGCTAGGCAA	SEQ ID NO.19	-	NS5B 8811-8830

RNAi reagent	The RNAi sequence ^#	SEQ ID NO.	The Luc-HCV reporter plasmid	The HCV position
RNAi reagent	The RNAi sequence ^#	SEQ ID NO.	The Luc-HCV reporter plasmid	The HCV position	C-10	gTCCACAGTTACTCTCCAGG	SEQ ID NO.20	-	NS5B 9019-9037
C-11	gCCTCTTCAACTGGGCAGTA	SEQ ID NO.21	-	NS5B 9170-9188	C-10	gTCCACAGTTACTCTCCAGG	SEQ ID NO.20	-	NS5B 9019-9037
C-11	gCCTCTTCAACTGGGCAGTA	SEQ ID NO.21	-	NS5B 9170-9188	C-12	AGCTTAAACTCACTCCAAT	SEQ ID NO.22	-	NS5B 9196-9214
3′-1	GCTCCATCTTAGCCCTAGT	SEQ ID NO.23	19	5-23 ^*	C-12	AGCTTAAACTCACTCCAAT	SEQ ID NO.22	-	NS5B 9196-9214
3′-1	GCTCCATCTTAGCCCTAGT	SEQ ID NO.23	19	5-23 ^*	3′-2	gTCCATCTTAGCCCTAGTCA	SEQ ID NO.24	19	7-25 ^*
3′-3	GTCACGGCTAGCTGTGAAA	SEQ ID NO.25	19	22-40 ^*	3′-2	gTCCATCTTAGCCCTAGTCA	SEQ ID NO.24	19	7-25 ^*
3′-3	GTCACGGCTAGCTGTGAAA	SEQ ID NO.25	19	22-40 ^*	3′-4	ACGGCTAGCTGTGAAAGGT	SEQ ID NO.26	19	25-43 ^*
3′-5	GCTGTGAAAGGTCCGTGAG	SEQ ID NO.27	19	32-50 ^*	3′-4	ACGGCTAGCTGTGAAAGGT	SEQ ID NO.26	19	25-43 ^*
3′-5	GCTGTGAAAGGTCCGTGAG	SEQ ID NO.27	19	32-50 ^*	3′-6	GGTCCGTGAGCCGCATGAC	SEQ ID NO.28	-	41-59 ^*^
3′-7	GCCGCATGACTGCAGAGAGT	SEQ ID NO.29	-	50-69 ^*^	3′-6	GGTCCGTGAGCCGCATGAC	SEQ ID NO.28	-	41-59 ^*^
3′-7	GCCGCATGACTGCAGAGAGT	SEQ ID NO.29	-	50-69 ^*^	3′-8	ACTGGCCTCTCTGCAGATCA	SEQ ID NO.30	-	76-95 ^*^

The small letters representative is not duplicated certainly or the genomic sequence of HCV corresponding to the HCV fusion

Table 3 luciferase HCV fusion plasmid

Luciferase-HCV fusion plasmid	The HCV target region
Luciferase-HCV fusion plasmid	The HCV target region	#
20	5 ' 1-is to-5 ' 5	#
20	5 ' 1-is to-5 ' 5	#16	5 ' 3-is to-5 ' 10
#17	5 ' 6-is to-5 ' 10	#16	5 ' 3-is to-5 ' 10
#17	5 ' 6-is to-5 ' 10	#12	5 ' 7-is to-5 ' 10, coding region-1
#18	Coding region-3	#12	5 ' 7-is to-5 ' 10, coding region-1
#18	Coding region-3	#9	3 ' 1-is to-3 ' 8
C2&4	Coding region-2, coding region-4	#9	3 ' 1-is to-3 ' 8
C2&4	Coding region-2, coding region-4	C5	Coding region-5
C6	Coding region-6	C5	Coding region-5
C6	Coding region-6	C7	Coding region-7
C8	Coding region-8	C7	Coding region-7
C8	Coding region-8	C9	Coding region-9
C10	Coding region-10	C9	Coding region-9
C10	Coding region-10	C11&12	Coding region-11, coding region-12
C6-C9-C12-3’1	Coding region-6, coding region-9, coding region-12,3 ' 1	C11&12	Coding region-11, coding region-12

In order to check the effect of selected RNAi sequence, directly RNAi reagent is delivered to culturing cell with luciferase HCV fusion plasmid.The synoptic diagram of luciferase HCV fusion plasmid that representative is used for these experiments is presented at the left side picture frame of figure.It includes the gene order that a coding is fused to the fluorescence luciferin zymoprotein of 100bp nucleic acid stretching area, and--RNAi reagent originates from this HCV zone--is corresponding with the HCV target sequence.Directly at the sequence RNA i reagent of 100bp intra-zone will, if effectively, degraded luciferase HCV transcription product, thereby reduce or eliminate luciferase expression.Table 3 has been enumerated the HCV target area of some corresponding luciferase HCV fusion plasmids and use.

Pre-synthetic siRNA reagent is that (Lafayette CO) obtains from Dharmacon limited-liability company.The Huh7 cell be before transfection 24 hours with every hole 9.5 * 10 ⁵The density of individual cell is seeded on 12 well culture plates.The cell of 30-40% is to merge in transfection.(VennNova, Pompano Beach FL), and at room temperature cultivated 1 hour to be mixed into the NovaFECTOR of 15 μ l in 350 μ lOptiMEM (InvitrogenInc.) substratum.In 50 μ l OptiMEM, be mixed into 0.05 μ g pRL-SV40 (Promega), the Luc-HCV reporter plasmid of 0.45 μ g, and the suitable R NAi reagent of 2 μ l (amount of μ M).NovaFECTOR solution is joined in the DNA/RNAi mixture, and at room temperature cultivated 15 minutes.With OptiMEM the cell cleaning once and with 400 μ l NovaFECTOR/DNA/RNAi mixtures is carried out transfection.Then cell is contained 5%CO at 370C ₂Air conditions was cultivated 1.5 hours down.The perfect medium of a milliliter is joined in each hole, and continue to cultivate other 2.5 hours, replace with fresh perfect medium at substratum at that time.Further cultivate continuity two days.

After two days, the sucking-off substratum and with cytolysis and according to the dual luciferase working specification of producer (Promega Madison WI) measures the expression of luciferase.The light unit (RLUs) relevant based on the normalizing luciferase calculates with respect to the inhibition percentage with the non-specific RNAi kind cells transfected that does not have known interior living target.According to the transfection efficiency difference of expressing based on the Renilla luciferase activity data are carried out stdn, wherein the Renilla luciferase comes from the pRL-SV40 plasmid with luciferase HCV fusion plasmid cotransfection.

Figure 12 shows the result by the luciferase expression inhibition of relative light unit metering, wherein by using different RNAi reagent target to five different HCV100bp zones; And the result that the luciferase expression that shows Figure 13 suppresses the wherein data of Figure 12 is represented as percent value.By observing these results, notice at the RNAi reagent of all selections and realize 50% inhibition at least, and the plasmid that surpasses half has reached more than 80% the inhibition level of luciferase expression.

Figure 14 demonstration is used for checking the reproducibility of target to the experimental result of the multiple segmental four kinds of different RNA i reagent of a 100bp sequence in HCV5 ' zone (5 '-1,5 '-2,5 '-3 and 5 '-4).Should arrive and note each reagent test and the renaturation of overstating is fabulous.

Figure 15 shows at target multiple segmental five different RNAi reagent (5 '-3,5 '-4,5 '-5,5 '-6 and 5 '-7) transfection 24 and change of luciferase expression inhibition per-cent after 48 hours to the 100bp sequence in HCV5 ' zone.

Figure 16 shows at target multiple segmental two different RNAi reagent (5 '-1 and 5 '-3), target different segmental five different RNAi reagent (3 '-1 to the 100bp sequence in HCV3 ' zone to the 100bp sequence in HCV5 ' zone, 3 '-2,3 '-3,3 '-4 and 3 '-5) and one be directed to the RNAi reagent transfection 44 of HCV opening code-reading frame zone 100bp sequence and luciferase expression suppresses per-cent after 72 hours change.

Figure 18 shows because the luciferase inhibition that causes with target of all kinds HCV genome different zones RNAi agent treated to the luciferase HCV fusion plasmid.Luciferase activity is with the measurement to huh7 cell 48 hours of RNAi reagent cotransfection.Can from these data, find out RNAi reagent effectively target HCV genome all the zone and cause luciferase report signal intensive is suppressed.

Embodiment 3B selection and check are at the RNAi reagent of luciferase HCV replicon system

Though understood many single steps that HCV duplicates, still do not have up to date to find can transmitted virus the tissue culture system of life cycle, make to the virus research difficulty that becomes.Yet, developed an external replicon system (referring to for example, U.S. Patent No. 5,585,258; 6,472,180; With 6,127,116 to Rice, etc.).Replicon be can comprise be used to select with the self-replicating of the HCV geneome RNA of the marker gene of checking copying partly.After through the step in the infectious cycle, duplicate needed suitable virus protein by the mechanical translation RNA and the producer gene group of cell, and selected marker, if present.Produce total length and subgenomic replicon and prove function though only nonstructural proteins is essential.The self-replicating characteristic of RNA has kept not relying on the expression of structure gene.Even when being present in when expressing in the genomic replicon of total length HCV, core and envelope protein matter are failed packaging gene group effectively to the infectivity particle--cause being used to the disappearance studying the virus packing, overflow and enter the model system of step again.In any case replicon can be recreated a biological phenomena and the machine-processed part that HCV utilizes.

Except utilize luciferase or other such report thing as an alternative, the active level of replicon can be regulated according to multiple other method.The inhibition that HCV is duplicated can be estimated by the level relatively of observation nonstructural proteins, and wherein proteinic level relatively is to detect by the immunofluorescence microscopy of having utilized the culture plate with HCV monoclonal antibody specific that commerce can get.Additionally or in addition, Q-PCR can be used to measure the level relatively from the HCV geneome RNA of each transfection situation.

The performance of siRNA reagent suppressor gene group rna replicon is to check by measurement Lei Niliya luciferase expression in by the clone of luciferase HCV fused replicon transformation.It is because lack corresponding sequence and comprised into current check as just nonspecific contrast that five siRNAs can not test out in the sub-genome duplication subsystem.The synoptic diagram of luciferase HCV replicon is presented among Fig. 8 B and the 8C.In cell inoculation to 96 well culture plate; Cultivate through 24 hours, interferon alpha 2B (IFN), known can suppress HCV replicon activity (Blight, etc. Science.290:1972-74 (2000)), the concentration with every milliliter of 100 unit is added in the specific hole.Then carry out cultivating in other 48 hours, abandon substratum and produced cell extract in position and then measured critically luciferase activity (data do not show) corresponding to luciferase mRNA level.In check, comprise 29 ∑ cells of luciferase HCV replicon (in Fig. 8 B, showing) with target RNAi reagent transfection in a plurality of zones to the HCV genome to RNAi reagent.The level relatively of harvested cell, generation extract and assessment luciferase activity after 48 hours.Figure 19 has shown the result of the luciferase inhibition that produces by the siRNA reagent that points to luciferase HCV replicon different zones.The RNAi reagent of sensing from C-1 to the C-5 coding region does not have the target luciferase HCV replicon and has served as additional nonspecific contrast.Similarly the target of All Ranges can be used as effective inhibition site at siRNA reagent in the HCV genome.

In case selected several high functionalized RNAi and, with that their triple transfections entered the cell that carries the replicon system respectively through check.A parallel control has comprised the uncorrelated RNAi kind of transfection respective numbers.Compared with respect to one group by the triple transfections for the parallel plate of RNAi kind transfection only to active inhibition.

Three RNAi reagent are proved, and are that long complementary self-annealing oligonucleotide special site that generate and that be cloned into triple promotor AAV carriers is arranged at each those encoding sequence of corresponding shRNA.Cause the method for the highest transduction efficiency of liver organization system packed these constructs then according to the utilization of here describing and enter virion.The total length of the promotor of each triple promotor box/RNAi/ terminator component is little (about 400 Nucleotide); Three promotor/RNAi/ components of common connection have produced a sequence that 1200-1300 Nucleotide is long, far below the top size restriction from complementary AAV.

The inhibition activity of these particles is to test carrying on the clone of replicon.Triple promoter constructs of expressing three incoherent shRNA kinds are as negative control.Effect by above-mentioned analytical technology monitoring shRNA sequence.

Embodiment 4: the exploitation of triple promoter expression constructs

Make up triple promoter expression constructs and comprise that three drive independent startup and the terminator sequence that single shRNA kind is expressed on similar abundance level.The repetition of promoter element can be permitted the expression cassette to the integration of recombination event sensitivity; Thereby the promotor of three uniquenesses and terminator are differentiated out and are used.The synthetic of small nuclear rna s and transfer RNA (tRNA) s instructed by the rna plymerase iii (pol III) under the domination of polIII specificity promoter.Owing to instructed by these controlling elements to have produced relatively abundanter transcript, pol III promotor comprises deriving from U6 and H1 gene, be used to drive shRNA expression (referring to for example, Domitrovichand Kunkel. Nucl.Acids Res.31 (9): 2344-52 (2003); Boden, etc. Nucl.Acids Res.31 (17): 5033-38 (2003a); And Kawasaki, etc. Nucleic Acids Res.31 (2): 700-7 (2003)).

At first, in comprising single, the specific promotor carrier of (in Fig. 7, showing), carried out assessment to the relative promotor intensity of pol III specific sequence.Each promoter construct driven those demonstrated to luciferase activity have the shRNA of functional inhibition expression (Elbashir, etc. Nature.411:494-498 (2001 a)).Since there is lot of data to show the successful Application of the U6 promotor that shRNA is expressed, it can be used as the standard of estimating other promotor relative intensity.Most of certified promotors are quite short, and most length is in 200-300 Nucleotide scope.Long overlapping oligonucleotide be used to ressemble promotor and terminator and then the clone enter in the multiple clone site of shRNA flank.Promotor is supporting with the termination signal that is present in gene downstream, promotor source naturally.

The report thing that commerce by cotransfection can get--pGL3 contrast (in Fig. 7, showing) or pRLSV40 (Promega, Madison, WI Madison, activity WI) reduces the relative intensity in each promotor of extracorporeal evaluate.The utilization standard technique will check and report that the construct transfection enters permissive cell.The functional shRNA that contrast is made up of wherein at luciferase the check promoter construct is substituted by incoherent shRNA sequence.The 3rd construct of human secreted protein alpha-1 antitrypsin (hAAT) of encoding is transfected in the cell so that estimate variation in transfection efficiency.In order to assess the inhibition activity of shRNA, utilize commercial reagents box (Promega, Madison, WI) the measurement luciferase activity of standard.The luciferase expression of shRNA regulation and control reduces, and is normalized into the hAAT level, is the indirect measurement to promotor strength.Additionally or in addition, to carry out quantitative PCR in real time analysis (Q-PCR) from parallel laboratory test culture plate results and purified RNA at the luciferase rna level.

In case it is right to identify suitable promotor and terminator, just can design last triple promotor RNAi expression cassettes.Several designs of last carrier have been checked, be included in to have all three promotors in the arranged in series or reach in the clockwise direction configuration (for example, being transcribed) in the clockwise direction and have all three promotors or any variant wherein from the top of box DNA and bottom chain.Three such configurations show in Fig. 3 A, 3B and 3C.

The configuration that shows in Fig. 3 B and 3C is transfected to be entered in the cell and utilizes the luciferase activity analytical study it suppresses active.The distinct rna i kind of two or more promoters driven can cause the additional or collaborative effect that suppresses, thereby, in order to be evaluated at the functional and relative intensity of each promotor in the triple promoter expression constructs of this paper, produce multiple expression cassette and detailed description in table 4.Use these kinds, the inhibition effect of the shRNA of each promoters driven of threefold representation inside according to luciferase analysis measure.Additionally, utilize the relatively level of Q-PCR assessment by the transcript of each promoters driven.Though hairpin RNA usually can stop from the complementary person's character these rna transcriptions direct Q-PCR is originally measured, three different intimate onesize non-hair clip transcripts can insert carrier to replace using the shRNA of viral multiple promoter RNAi expression construct, and wherein viral multiple promoter RNAi expression construct has such as the box in Fig. 3 D and 3E demonstration.

3 type promotor: U6-1, U6-8, U6-9, long H1, human Y4, the human Y5 of following RNA Pol III, selected, synthetic and clone enter single or the multiple promoter construct in.Then luciferase specificity shRNA is cloned into the downstream of a certain promotor in the downstream of single promoter construct or the triple promoter construct (table 4).

After transfection 72 hours, (Promega, Madison WI) measure luciferase expression with the dual luciferase working specification according to producer for sucking-off substratum and dissolved cell.The cell (negative control) of comparative simulation treatment calculates the inhibition percentage based on the relevant light unit (RLUs) of standardized luciferase.An incoherent RNAi reagent is used as simulation/negative control in each experiment.Carry out luciferase RLUs stdn according to the Lei Niliya luciferase that goes out with the pRL-SV40 plasmid expression of target plasmid co-transfection at transfection efficiency.

In the construct of 293 cells (17B) shown in Huh7 cell (17A) construct shown in Fig. 3 C and Fig. 3 B, all promotors demonstrate similar activity.The rejection characteristic that can find out the shRNA in single and multiple promoter scope is similar.Data presentation shRNA is suppressed in triple promotor scopes and is similar in Huh7 and 293 cells.

Table 4: the promotor/shRNA that is used for assessing in the construct type shown in inhibiting relatively Fig. 3 B of each expression cassette and the 3C inserts.

Construct	Promotor/shRNA
	Promotor/shRNA			A U6-1	B U6-8	C U6-9
	Construct I	shRNA-LUC	Empty	A U6-1	B U6-8	C U6-9	Empty
Construct II	Construct I	shRNA-LUC	Empty	Empty	shRNA-LUC	Empty	Empty
Construct II	Construct III	Empty	Empty	Empty	shRNA-LUC	Empty	shRNA-LUC

The plasmid that includes the promotor/shRNA/ box type that shows among Fig. 3 B is used to assess the relative restraining effect of the expressed shRNA reagent that goes out of multiple promoter expression cassettes.Including target to the plasmid of the shRNA construct of HCV genome different zones operationally is placed under the domination of different promoters.Table 5 is presented at shRNA under the domination of independent promotor, is included in the shRNA reagent of (inactivation just) promotor that is positioned at that B order under controlling.Include from being connected to 5 of luciferase genes '-3 and enter in the Huh7 cell to the luciferase HCV fusion plasmid #16 of the HCV sequence in 5 '-10 zones and multiple promoter construct cotransfection together.Figure 20 show when and multiple promoter/pair shRNA expression construct rather than during with the construct cotransfection that includes the single shRNA after being connected to single promotor the inhibition to luciferase increased.

Table 5 shows in Fig. 3 B is used for being evaluated at promotor/shRNA inset in the single relative inhibiting construct type with double starting of multiple promoter expression cassettes.

Construct	Promotor/shRNA
	Promotor/shRNA			A U6-9	The B inactivation	C U6-8
	Construct I	5-’3	5’-1	A U6-9	The B inactivation	C U6-8	Empty
Construct II	Construct I	5-’3	5’-1	5-’3	C-12	Empty	Empty
Construct II	Construct III	5-’3	5’-8	5-’3	C-12	Empty	Empty
Construct IV	Construct III	5-’3	5’-8	5-’3	5’-8	5’-6	Empty

The external check of the triple promoter constructs of embodiment 4B:shRNA

The triple promotor boxes that show at Fig. 3 C are to use following promotor to make: U6-9 is in the A site, and U6-1 is in the B site, and U6-8 is in the C site.Promoters driven target a plurality of positions to the HCV genome the shRNA sequence transcribe or the promotor heel with in " sky " T ' s stretching area in the configuration to prevent reading over of promotor.Single promotor/shRNA construct in contrast utilizes the U6-1 promotor to make up.Single or triple promoter constructs are with the luciferase HCV reporter plasmid cotransfection that comprises different HCV target area.Figure 21 zone that to have shown that to use the result that luciferase suppresses behind single or the multiple promoter construct cotransfection, wherein single or multiple promoter construct be target to the HCV genome different and the luciferase HCV fusion plasmid of thrin are comprising the sequence that comes from HCV genome target region.Cotransfection to Huh7 cell was measured luciferase activity after 72 hours.The shRNA that can see the C-12 zone of the special HCV of being directed in the chart of the top of Figure 21 demonstrates has suitable inhibition activity to the luciferase reporter plasmid that comprises C-12 coding region among the HCV.When specificity at other regional shRNA of HCV by individually or as the part of multiple promoter box of the present invention and do not observe specific inhibition when expressing.Similar result can observe in middle plot, and wherein luciferase HCV reporter plasmid has comprised the sequence from C-9 coding region among the HCV.In this example, the shRNA that is directed to the C-9 zone specifically that expresses from single promotor or triple promotor box has intensive and suppresses active in the shRNA reagent that had detected.Bottom pictorialization used single or triple promoter constructs and comprises the luciferase that causes behind the reporter plasmid cotransfection in HCV5 ' 6 zones and suppress.Can see that intensive suppresses the result and comes from and comprise the shRNA that specificity is directed to 5 ' 6 targets in single or the multiple promoter construct.Triple promoter construct of the present invention is used for suppressing effectively the specific gene target.

Embodiment 5; Check in the body of the triple promoter construct reagent of shRNA

Assessment to multiple promoter construct of the present invention is by hydrodynamic tail vein injection program in vivo, and multiple promoter/shRNA plasmid DNA that shows among use Fig. 3 and suitable Lampyridea luciferase HCV merge reporter plasmid cotransfection mouse liver and carry out.Multiple promoter used herein/shRNA plasmid has controlled that target encodes 9 to the HCV, the expression of the shRNA kind of coding 12 and 5 ' 8 positions.Operation report construct and incoherent shRNA injection negative control mouse.In addition, use the plasmid injection mouse of expressing the Lei Niliya luciferase.This protein is used to the mouse liver transformation efficiency is carried out stdn.Injected back 48 hours, and put to death mouse, collect liver.Use Promega luciferase test kit to detect the Lampyridea luciferase activity and the Lei Niliya luciferase activity of liver lysate.The assessment of comparison negative control is expressed institute's inductive inhibition level from the shRNA of hair clip construct.Result in the table 6 has shown that triple promoter construct of the present invention has suppressed report signal in vivo effectively.

The triple promotors of table 6/shRNA plasmid is to the inhibition per-cent of Lampyridea luciferase signal

Group #	n	Plasmid expression shRNA (5ug/ mouse)	Reporter plasmid (12ug/ mouse)	Luciferin RLU (being standardized as Renilla)	Compare the inhibition % of triple shRNA with non-specific shRNA contrast
Group #	n	Plasmid expression shRNA (5ug/ mouse)	Reporter plasmid (12ug/ mouse)	Luciferin RLU (being standardized as Renilla)		21	5	Triple promotor/shRNA (5 ' 8as, C-9s, C-12as)	C-9 (pBen71)	0.05	98％
23	5	Single non-specific 5 '-3 (non-specific shRNA)	C-9 (pBen71)	3.20	n/a	21	5	Triple promotor/shRNA (5 ' 8as, C-9s, C-12as)	C-9 (pBen71)	0.05	98％
23	5	Single non-specific 5 '-3 (non-specific shRNA)	C-9 (pBen71)	3.20	n/a	25	5	Triple promotor/shRNA (5 ' 8as, C-9s, C-12as)	C-11/12 (pBen73)	1.00	93％
27	5	Single non-specific 5 '-3 (non-specific shRNA)	C-11/12 (pBen73)	15.10	n/a	25	5	Triple promotor/shRNA (5 ' 8as, C-9s, C-12as)	C-11/12 (pBen73)	1.00	93％

To include by the injection of tail vein injection or hepatic vein and to express target to the infectious AVV particle of the carrier of HCV sequence and be delivered to normal mouse.The infectious AVV particle of expressing three uncorrelated shRNAs is as negative control.Originally, used a quite high viral dosage, for example 2 * 10 ¹²The vector gene group, though follow-up experiment is used to set up dose-response curve.Using a suitable Lampyridea luciferase HCV to merge reporter plasmid by hydrodynamic tail vein injection program at different time injects.After injection reporter plasmid 48-72 hour, put to death mouse, collect liver and serum sample.Use the Lampyridea luciferase activity to assess the efficient of the shRNA that AVV sends as benchmark.In addition, the liver level of the serum level of utilization monitoring hAAT or Lei Niliya luciferase decides the transfection efficiency between animal.Measuring the serum level of liver enzyme alanine aminotransferase, aspartic transaminase and tumor necrosis factor alpha can not induced by treatment to guarantee overall hepatotoxicity.

Embodiment 6: the shRNA at the HBV model system of duplicating in the body that check AAV sends

Do not exist ideal to can be used for checking that AAV sends at the small animal model of the shRNA expression construct effect of HCV.Yet, can be used for assessing the inhibition degree of liver to virus replication to the check of replacing the AAV expressed rna i construct in the model system at one.Though it must be different that the sequence of the shRNA that is sent is formed at such model, the rest part of this system comprises triple promoter expression vectors of AAV and packing composition, is keeping not changing.Used model system at hepatitis B virus (HBV).Be included in triple promotor AAV expression construct with target to the shRNA sequence of HBV be according to the effect of the shRNA sequence of announcing up to now select (McCaffrey, etc. Nature Biotech.21 (6): 639-644 (2003); Ying, etc. Biochem.Biophvs.Res.Commun.309 (2): 482-484 (2003); Klein, etc. Gastroent.125 (1): 9-18 (2003); And Shlomai, etc. Hepatoloav.37 (4): 764-70 (2003)).Suitable AAV expression construct with three HBV specificitys of triple promoters driven shRNAs enters in the virion according to the method packing of here describing then.

By hydrodynamic transfection program mouse is carried out the injection first time with the expression plasmid that carries the HBV genome sequence and allow to set up hbv replication.In order to assess transfection efficiency, the plasmid of a coding hAAT is added into and is used for common injection in the mixture.The active initial index of HBV is to use the enzyme-linked immunosorbent assay analysis to collect and assesses from the hepatitis B virus surface antigen (HBsAg) hemorrhage, that occur by the serum the treatment animal of posterior orbit neuroplexus and HBV cAg (HBcAg).

Establish in mouse liver after the duplicating of HBV, the AAV virion of having packed triple promoter constructs of shRNAs among the coding HBV utilizes tail vein injection or hepatic vein injection and is introduced in the mouse.Use carries the AAV virion of AAV expression construct of three uncorrelated shRNAs of HBV of coding as negative control.At the terminal point of experiment, the downward modulation of HBsAg and HBcAg protein quantity in the monitoring serum sample.Additionally or in addition, the level relatively by HBV RNA in the Q-PCR assessment liver organization.Monitor the hepatotoxicity that alanine aminotransferase, aspartic transaminase and tumor necrosis factor alpha in the serum come evaluation system by ELISA.

Figure 10 is the graphic extension of the triple promotor box of a precursor embodiment, and the triple promotor boxes of this precursor have specific restriction site and can be used for inserting or removing RNAi kind or terminator element or be used to cut off U6-9, pU6 or the U6-8 promotor that provides.In addition, arrow shows the transcriptional orientation of each three promotor/RNAi/ terminator component.Figure 11 A and 11B/11C have shown the nucleotide sequence (being respectively SEQID NO 31 and 32) in two embodiments of precursor promotor box of the present invention.Figure 10, the position of demonstration U6-9, pU6 or U6-8 promotor, and the restriction site of precursor promotor box of the present invention.

Though the present invention is described with reference to specific embodiment, it should be understood that and can make different changes to those skilled in the art and utilize equivalent to substitute within the real spirit and scope of the present invention.In addition, in order to realize that purpose of the present invention, spirit and scope can adopt many modifications to adapt to certain location, material or operation.The modification of all these classes is all within the scope of the invention.

Whole reference of here quoting are in order to help the understanding of the present invention, and they are not any limitation of the invention.

Sequence table

<110>Benitec，Inc.

Roelvink，Petrus W.

Suhy，David A.

Kolykhalov，Alexander A.

＜120〉be used for sending simultaneously the multiple promoter expression cassettes of RNAi reagent

<130>BENI/0003

<150>US 60/553,920

<151>2004-03-17

<150>US 60/550,504

<151>2004-03-05

<160>32

<170>PatentIn version 3.3

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cgagccttgt gatgagttcc gttgcatgaa attctcccaa aggctccaag atggacagga 900

aagggcgcgg ttcggtcacc gtaagtagaa taggtgaaag actcccgtgc cttataaggc 960

ctgtgggtga cttcttgcta gcgaccttac gtgttcatgg aattctgtac cgtatatagc 1020

atgactgcgg ccgccaattc atctatgtcg ggtgcggaga aagaggtaat gaaatggcat 1080

tatgggtatt atgggtctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 1140

tattgggcgc tctt 1154

<210>32

<211>1971

<212>DNA

＜213〉artificial sequence

<220>

＜223〉multiple representation of RNAi

<400>32

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtaactataa 60

cggtcctaag gtagcgaaag ctcagatccg cggccgcgta gtacgatgac tagcatgcag 120

ggcggtgcgg ctcaggctct gccccgcctc cggggctatt tgcatacgac catttccagt 180

aattcccagc agccaccgta gctatatttg gtagaacaac gagcactttc tcaactccag 240

tcaataacta cgttagttgc attacacatt gggctaatat aaatagaggt taaatctcta 300

ggtcatttaa gagaagtcgg cctatgtgta cagacatttg ttccaggggc tttaaatagc 360

tggtggtgga actcaactag tgtagatttt tttctgcagg catagcagag atctgttcgg 420

ctttacgtca cgcgagggcg gcagggagga cggaatggcg gggtttgggg tgggtccctc 480

ctcgggggag ccctgggaaa agaggactgc gtgtgggaag agaaggtgga aatggcgttt 540

tggttgacat gtgccgcctg cgagcgtgct gcggggaggg gccgagggca gattcgggaa 600

tgatggcgcg gggtgggggc gtgggggctt tctcgggaga ggcccttccc tggaagtttg 660

gggtgcgatg gtgaggttct cggggcacct ctggaggggc ctcggcacgg aaagcgacca 720

cctgggaggg cgtgtgggga ccaggttttg cctttagttt tgcacacact gtagttcatc 780

tttatggaga tgctcatggc ctcattgaag ccccacggat ctgggcagga agagggccta 840

tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga 900

attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa 960

tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc 1020

gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaggatcc 1080

ggttattttt ttcaattgta cagctctggt agcggtaacc atgcgtattt gacacacgaa 1140

ggaactaggg aaaaggcatt aggtcatttc aagccgaaat tcacatgtgc tagaatccag 1200

attccatgct gaccgatgcc ceaggatata gaaaatgaga atctggtcct taccttcaag 1260

aacattctta accgtaatca gcctctggta tcttagctcc accctcactg gttttttctt 1320

gtttgttgaa ccggccaagc tgctggcctc cctcctcaac cgttctgatc atgcttgcta 1380

aaatagtcaa aaccccggcc agttaaatat gctttagcct gctttattat gattattttt 1440

gttgttttgg caatgacctg gctacctgtt gtttctccca ctaaaacttt ttaagggcag 1500

ggaattgatc tagaaaaaaa aaagctagtg gtaccggtcc tacgcggggc cctttaccca 1560

gggtgccccg ggcgctcatt tgcatgtccc acccaacagg taaacctgac aggtcatcgc 1620

ggceaggtac gacctggcgg tcagagcacc aaacatacga gccttgtgat gagttccgtt 1680

gcatgaaatt ctcccaaagg ctccaagatg gacaggaaag ggcgcggttc ggtcaccgta 1740

agtagaatag gtgaaagact cccgtgcctt ataaggcctg tgggtgactt cttgctagcg 1800

accttacgtg ttcatggaat tctgtaccgt atatagcatg actgcggccg ccaattcatc 1860

tatgtcgggt gcggagaaag aggtaatgaa atggcattat gggtattatg ggtctgcatt 1920

aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct t 1971

Claims

1. the genetic constructs that comprises multiple promoter expression cassettes, described multiple promoter expression cassettes comprises at least three promotors/RNAi/ terminator component, each promotor/RNAi/ terminator component RNAi kind of comprising promoter element, terminator element and being operably connected wherein with promoter element and terminator element, and wherein each RNAi kind has nothing in common with each other each other.

2. according to the genetic constructs of claim 1, wherein genetic constructs also comprises this construct packing is entered necessary element in the infectious viral particle.

3. according to the genetic constructs of claim 1, wherein the sequence of each the terminator element in each promotor/RNAi/ terminator component has nothing in common with each other each other.

4. according to the genetic constructs of claim 1, wherein the sequence of each promoter element in each promotor/RNAi/ terminator component has nothing in common with each other each other.

5. according to the genetic constructs of claim 1, wherein the sequence target of RNAi kind has the gene of sequence single nucleotide polymorphism (SNP) between the variant to those, wherein each RNAi kind can target to the hypotype of one or more variant.

6. according to the genetic constructs of claim 1, wherein the sequence target of RNAi kind experiences the virus sequence of sudden change fast.

7. according to the genetic constructs of claim 1, one or more variants of the sequence target gene of each RNAi kind wherein.

8. method that is suppressed at the nucleic acid target level of expressing in the cell, be included under the condition that nucleic acid target to be finished is enough to express genetic constructs and cells contacting with claim 1, wherein each RNAi kind is identical with the sequence of this nucleic acid target basically or the variant with described nucleic acid target is identical basically.

9. method that is modified at one or more nucleic acid target of expressing in the cell comprises:

A) the genetic constructs packing with claim 1 enters virion;

B) this virion is delivered to cell;

C) under the condition that nucleic acid target to be finished is enough to express, express from three or more RNAi kind in the multiple promoter expression cassettes of claim 1.

10. according to the method for claim 9, wherein one or more nucleic acid target basically with cause or to keep the necessary gene of morbid state identical.

11. according to the method for claim 10, wherein said morbid state is a cancer.

12. according to the method for claim 9, wherein said nucleic acid target is identical with the gene of finding in virus basically.

13. according to the method for claim 12, wherein said virus is hepatitis C virus.

14. according to the method for claim 12, wherein the sequence of three or more RNAi kind is identical with the gene of finding in this virus basically and basically with identical as two or more sudden changes of escaping in the described virogene that mutant strain produces.

15. a method that is modified at one or more nucleic acid target of expressing in the cell comprises:

A) the genetic constructs packing with claim 1 enters non-virion;

B) should be delivered to cell by non-virion;

C) under the condition that nucleic acid target to be finished is enough to express, express three or more RNAi kind from the multiple promoter expression cassettes of claim 1.

16. according to the method for claim 15, wherein said nucleic acid target basically with cause or to keep the necessary gene of morbid state identical.

17. according to the method for claim 15, wherein said nucleic acid target is identical with the gene of finding in virus basically.

18. according to the method for claim 17, wherein said virus is hepatitis C virus.

19. according to the method for claim 18, wherein the sequence of three or more RNAi reagent is identical with the gene of finding in this virus basically and basically with identical as two or more sudden changes of escaping in the described gene that mutant strain produces.

20. the method for one or more nucleic acid target that a processing is expressed in zooblast, tissue or organ, described method comprises that the genetic constructs that will contain the claim 1 of any one nucleotide sequence among the SEQ ID NO:1-30 imports described zooblast, tissue or organ.