CN101052422A - Compositions and methods for protein production - Google Patents

Abstract

Disclosed herein are methods and compositions for enhanced protein production and overexpression using engineered zinc finger proteins.

Description

The compositions and the method that are used for protein production

The cross reference of related application

The application requires the priority of U.S. Provisional Patent Application number 60/610,853 (JIUYUE was submitted on the 16th in 2004) and 60/661,841 (submission on March 15th, 2005), includes its content in this paper in full and is used for all purposes.

About subsidize the rights statement of the invention of finishing under the research in federal government

Do not have.

Technical field

The invention belongs to the transcriptional regulatory field, especially protein expression and excess produce, for example extensive treatment protein that produces.

Background of invention

The controllable adjustment of transcribing can be used for research, diagnosis and treatment field.For example the transcriptional regulatory of the nucleotide sequence of coded protein can help producing in a large number be used for the treatment of, the recombiant protein of screening, lead optimization and target spot checking.

The feature of typical case's expression system is that the expression vector that host cell system comprises contains the allogeneic promoter that operability is connected in the cDNA of coding gene of interest product.A kind of such protein expression (and overexpression) system adopts SR α promoter, and this promoter is made up of SV40 early promoter and long terminal repetition R fragment and the part U5 sequence of human T-cell leukemia virus's 1 type.Takebe etc. (1988) Mol.Cell.Biol.8:466-472.Another overexpression system adopts human cytomegalic inclusion disease virus (CMV) immediate early promoter.United States Patent (USP) 5,168,062 and 5,385,839.These promoteres are regulated by the transcription factor of endogenous, natural generation all, and availability or the activity of these transcription factor in these systems can limit the amount of transcribing, thus limit protein matter output.And the overexpression of regulating the natural generation transcription factor of these promoteres can cause being subjected to usually the abnormal gene expression of these factors adjustings, may adverse effect be arranged to the expression of required gene outcome.

Other method that is used for protein production comprises regulates sequence with promoter or other to be incorporated into the gene that chromosome and expression regulated adjacent.Referring to for example United States Patent (USP) 5,272,071; 5,641,670; 5,733,761; 5,968,502 and 6,361,972.They depend on the effect of endogenous transcription factor simultaneously, therefore may be restricted, as above-mentioned SR α and CMV system.And they also are difficult to realize the chromosomal integration of the accurate targeting of exogenous polynucleotide.

The protein level of wanting to produce is higher than protein level that obtains with SR α and CMV promoter and the expression system that does not rely on the random integration of exogenous polynucleotide sequence.And, utilize transcription factor that the expression system of exogenous transcription factor can design customization, higher elasticity be provided and the potential of expressing the gene of interest product with higher level is provided.

Summary of the invention

In one aspect, provide and regulate the method that nucleotide sequence is transcribed in cell, this method is included in expresses the protein that is incorporated into the target site that comprises SEQ ID NO:1 in the cell, and wherein SEQ ID NO:1 operability is connected in nucleotide sequence.In some embodiments, a plurality of target site operability are connected in nucleotide sequence.

On the other hand, this paper has described and regulated the method that first and second nucleotide sequences are transcribed in cell, this method is included in expresses the protein that is incorporated into the target site that comprises SEQ ID NO:1 in the cell, wherein SEQ ID NO:1 operability is connected in first and second nucleotide sequences.A plurality of target site operability are connected in first nucleotide sequence, second nucleotide sequence or first and second nucleotide sequences.

In any method as herein described, protein can comprise, for example, and SEQ ID NO:25 or its equivalent (as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8).Therefore, this paper has described by express SEQ ID NO:25 or equivalent in cell and regulated the method that first (and/or second) nucleotide sequence is transcribed in cell.In some embodiments, this albumen also comprises transcription activating domain (as VP16).

In any method as herein described, the nucleotide sequence codified can be translated and produce one or more proteinic mRNA.And in any method as herein described, nucleotide sequence can comprise the cDNA sequence, for example one or more cDNA sequences of encoding antibody polypeptide (as heavy chain of antibody or light chain).In other embodiments, the nucleotide sequence codified is not translated into proteinic RNA molecule, for example siRNA, Microrna, rRNA, tRNA, snRNA or scRNA.

On the other hand, this paper has described and regulated the method that nucleotide sequence is transcribed in cell, this method comprises: express the protein that contains SEQ ID NO:25, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8 or equivalent in cell, wherein said protein bound is in target site; Wherein said target site operability is connected in nucleotide sequence.In some embodiments, described target site comprises SEQ ID NO:1.One or more target site operability can be connected in nucleotide sequence.In some embodiments, nucleotide sequence comprises the cDNA sequence.Protein also can comprise transcription activating domain, as the VP16 domain.

Aspect another, this paper has described the method for transcribing of regulating first and second nucleotide sequences in cell, this method comprises: express the protein that contains SEQ ID NO:25, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8 or equivalent in cell, wherein said protein bound is in target site; Wherein said target site operability is connected in first and second nucleotide sequences.In some embodiments, described target site comprises SEQ ID NO:1.One or more target site operability are connected in first and/or second nucleotide sequence.In some embodiments, described first and/or (second) nucleotide sequence comprise the cDNA sequence, the cDNA sequence of encoding antibody polypeptide (as light chain of antibody and/or heavy chain of antibody) for example.Described protein also can comprise transcription activating domain, as the VP16 domain.

Aspect another, provide the polypeptide that comprises SEQ ID NO:25.In some embodiments, this polypeptide comprises SEQ ID NO:2 or SEQ ID NO:7 or its equivalent.

Aspect another, the polynucleotide of any polypeptide described herein of encoding are provided.

Aspect another, provide the cell that comprises any polypeptide described herein and/or polynucleotide.

On the other hand, this paper provides the carrier that comprises SEQ ID NO:1; The cell that comprises the cell of any of these carrier and comprise one or more copies of the SEQ ID NO:1 that is integrated into cellular genome.

Those skilled in the art are understood that these and other embodiment by reading the disclosure.

Brief Description Of Drawings

Fig. 1 has shown the nucleotide sequence (SEQ ID NO:9) of hybridization SV40-R-U5 promoter (SR α promoter), and synthetic method is as described in the embodiment 1.The target site of representing embodiment 2 described various ZFP with underscore.

Fig. 2 has shown 2392/00 proteic aminoacid sequence (SEQ ID NO:10).This proteic domain is as follows.Aminoacid 3-9: nuclear localization sequence; Amino acid/11 5-109: Zinc finger domain; Amino acid/11 19-185:VP16 transcription activating domain; Amino acid/11 96-203:FLAG epi-position label.

Fig. 3 has shown coding 2392/00 proteic polynucleotide sequence (SEQ ID NO:11).The sequence part of this proteic each domain of encoding is as follows.Nucleotide 7-27: nuclear localization sequence; Nucleotide 43-327: Zinc finger domain; Nucleotide 355-555:VP16 transcription activating domain; Nucleotide 586-609:FLAG epi-position label.

Fig. 4 has shown 2392/10 proteic aminoacid sequence (SEQ ID NO:12).This proteic domain is as follows.Aminoacid 3-9: nuclear localization sequence; Amino acid/11 5-109: Zinc finger domain; Amino acid/11 19-185:VP16 transcription activating domain; Amino acid/11 96-203:FLAG epi-position label.

Fig. 5 has shown coding 2392/10 proteic polynucleotide sequence (SEQ ID NO:13).The sequence part of this proteic each domain of sign indicating number is as follows.Nucleotide 7-27: nuclear localization sequence; Nucleotide 43-327: Zinc finger domain; Nucleotide 355-555:VP16 transcription activating domain; Nucleotide 586-609:FLAG epi-position label.

Fig. 6 has shown the aminoacid sequence that refers to Zinc finger domain available from the double cross selective system with many three of identification target sequence GCTGTGGAA (SEQ ID NO:1).See embodiment 2 for details.

Fig. 7 has shown the level of immunoglobulin kappa chain mRNA in the cell that contains the integration transcript unit that comprises SR α promoter and κ chain cDNA.With coding VP16 activation domain (NVF) or be blended in the plasmid transfection cell of the 2393/00ZFP (2392-VP16) of VP16 activation domain.Numeral on the abscissa refers to the nanogram number of the DNA of transfection.See embodiment 3 for details.

Fig. 8 has shown that with the secretory antibody level in two kinds of cell lines (A and B) of the plasmid transfection of coding 2392/00 ZFP-VP16 fusion rotein (2392) what make comparisons is the cell of using the plasmid transfection of coding VP16 activation domain (NVF).GFP: the cell of using the plasmid transfection of encoding green fluorescent protein; Analogies: analogies cells transfected; Ntf: non-transfected cell.See embodiment 3 for details.

Fig. 9 has shown the level of immunoglobulin gamma heavy chain and immunoglobulin kappa light chain mRNA in the cell of the γ that contains amplification and κ chain cDNA, and γ and κ chain cDNA are in transcribe (" the high yield cell line " among the figure) under the control of SR α promoter.Result with the plasmid transfection cell of coding 2392/00-VP16 fusion rotein is " ZFP " on the transverse axis; The non-transfected cell cell is designated as " NT ".The mRNA level standardization is arrived the GAPDH level.See embodiment 3 for details.

Figure 10 has shown the level (being designated as " ZFP " among the figure) with the immunoglobulin G of the emiocytosis of the sequence stable transfection of coding 2392/00-VP16 fusion rotein.The IgG secretion level (being designated as " contrast ") that has also shown non-transfected cells.

Figure 11 has shown the relative IgG level with the cell of two kinds of different plasmid transfections of the transcript unit of the transcript unit of containing heavy chain-coding and light chain-coding.In a kind of plasmid, each transcript unit (is designated as SRa) transcribing under the control of SR α promoter among the figure.In another kind of plasmid, each transcript unit transcribes under the control SR α promoter, has added the SEQ ID NO:1 (being called SRa among the figure) of 8 additional copies on this promoter.2392/10-7 refers to the clone and separate thing with the Chinese hamster ovary celI of the nucleic acid stability transfection of coding 2392/10-VP16 fusion rotein.DG44 refers to the Chinese hamster ovary celI system of parent, untransfected.

Figure 12 has shown the green fluorescent protein mRNA level (being normalized into GAPDHmRNA) in the 2392/10-7 cell, and this cell contains the sequence of coding ZFP-VP16 fusion rotein.With containing the plasmid transfection cell that operability is connected in the various modification CMV promoteres of green fluorescent protein (GFP) coded sequence.The below of figure has shown the copy number of the target site SEQ ID NO:1 that inserts CMV promoter adjacent position in each construction, and the orientation of inserting target site.The rightmost side one coupled columns has shown the GFP mRNA level in the cell line that contains ZFP; Leftmost side post has shown the GFP mRNA level of the parental cell line of not expressing ZFP.

Figure 13 has shown with the different excretory erythropoietin of Epo expression constructs cells transfected (Epo) level.SR α Z6 refers to that Epo expresses the construction of the SR α promoter control that is contained seven SEQ ID NO:1 copies.CMV refers to that Epo expresses the construction that is subjected to the control of CMV promoter.CMVz10 refers to that Epo expresses the construction of the CMV promoter control that is contained 10 SEQ IDNO:1 copies.The rightmost side one coupled columns has shown the excretory Epo level of the cell line 2392/10-7 that contains ZFP; Leftmost side post has shown the excretory Epo level of the parental cell line of not expressing ZFP.See embodiment 9 for details.

Detailed Description Of The Invention

General introduction

Except as otherwise noted, this area is adopted in the enforcement of method, and the preparation of composition described herein and use Molecular biology known to the technical staff, biochemistry, chromatin Structure and analysis, chemistry, cell cultivate, The common technology of recombinant DNA and association area. This type of technology has been described in detail in detail in the document. Referring to such as Sambrook etc. " molecular cloning: laboratory manual " (MOLECULAR CLONING:A LABORATORY MANUAL), Second edition, Cold Spring Harbor Laboratory Press, 1989 and the third edition, 2001; Ausubel etc. are " new Partial numerator biological experiment guide " (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY), John Wiley ﹠ Sons, New York, 1987 and regular update; " Enzymology method " (METHODS IN ENZYMOLOGY) series, Academic Press, Santiago; Wolffe, " chromatin Structure and function " (CHROMATIN STRUCTURE AND FUNCTION), the third edition, Academic Press, Asia, the Holy Land The brother, 1998; " Enzymology method " (METHODS IN ENZYMOLOGY), the 304th volume, " chromatin " (Chromatin) (P.M.Wassarman and A.P.Wolffe compile), Academic Press, Santiago, 1999; " molecular biology method " (METHODS IN MOLECULAR BIOLOGY), the 119th volume, " dyeing The matter method " (Chromatin Protocols) (P.B.Becker volume) Humana Press, Totowa, 1999.

Definition

Term " nucleic acid ", " polynucleotides " and " oligonucleotides " are used interchangeably, and refer to wire or cyclic conformation Strand or double-stranded deoxynucleotide or ribonucleotide acid polymer, for the purpose of this disclosure, do not think these Term is limit by the length of polymer. This term can comprise the known homologue of natural nucleotide and base, sugar and/or Phosphoric acid part (such as the thiophosphate main chain) adorned nucleotides. Usually, the homologue of concrete nucleotides has identical The base pairing specificity, i.e. the homologue of A and T base pairing.

Term " polypeptide ", " peptide " and " protein " are used interchangeably, and refer to the amino acid residue polymer. This art Language also is applied to chemical homologue that wherein one or more amino acid are corresponding natural amino acids or modified derivative Amino acid polymer.

The technology of measuring nucleic acid and amino acid sequence homogeny is to know in the field. This type of technology generally comprises measures one The nucleotide sequence of individual gene mRNA and/or measure the amino acid sequence of a gene or mRNA encoding proteins, And with these sequences and another nucleotides or amino acid sequence comparison. Utilize this method also can measure and icp gene group order Row. Usually, homogeny refers to respectively accurate nucleotides-nucleotides or amino in two polynucleotides or the peptide sequence Acid-amino acid whose correspondence. Can more two or more (polynucleotides or amino acid) by detecting sequence homogeny percentage Sequence. Between two nucleic acid or amino acid aligned sequences accurately the pairing number multiply by 100 divided by shorter sequence length and obtain two The homogeny percentage of individual sequence. Smith and Waterman, " applied mathematics progress " (Advances in Applied Mathematics) local homology's algorithm of 2:482-489 (1981) provides the approximation ratio pair of nucleotide sequence. Utilize Dayhoff, " protein sequence and structure chart " (Atlas of Protein Sequences and Structure), M.O.Dayhoff compiles, 5 supplementary issues, and 3:353-358, national biomedical research foundation (National Biomedical Research Foundation), the Washington, DC special zone is developed and Gribskov,Nucl.Acids Res.14 (6): The standardized rating matrix of 6745-6763 (1986) can be with this algorithm application in nucleotide sequence. The science of heredity computer research Group (state of Wisconsin Madison) this algorithm of exemplary execution in " best fit (BestFit) " practical application is to measure Sequence homogeny percentage. Wisconsin sequence analysis software bag procedure manual the 8th edition (1995) (can be available from Wei Sikang Star state Madison science of heredity computer research group) default parameters of the method has been described. Determine identical in the present disclosure The property percentage method for optimizing be adopt the Edinburgh University all rights reserved, John F.Collins and Shane S. That Sturrok develops, IntelliGenetics, the MPSRCH program package of Inc. (Mountain View, California) distribution. Can use The Smith-Waterman algorithm of this software kit, (for example the breach opening penalizes 12 wherein default parameters to be used for grade form Divide, breach extend penalized 1 fen and breach 6 minutes). " Match " value has been reacted the sequence homogeny in institute's generated data. Those skilled in the art know other for the suitable procedure of homogeny between the sequence of calculation or similitude percentage, for example Another compares program BLAST (use default parameters). For example can adopt following default parameters BLASTN and BLASTP: genetic code=standard; Filter=do not have; Chain=two strands; Cutoff=60; Desired value=10; Matrix=BLOSUM62; Illustrate=50 sequences; Sorting=HIGH SCORE; Database=nonredundancy, GenBank+ EMBL+DDBJ+PDB+GenBank CDS translation+Swiss albumen+Spupdate+PIR. These journeys The visible following network address of the detailed description of order: http://www.ncbi.nlm.gov/cgi-bin/BLAST. As for sequence described herein, The required scope of sequence homogeny degree is about 20% to 100% and middle any integer value. Homogeny percentage between sequence Number generally is at least 70-75%, preferred 80-82%, and more preferably 85-90%, more preferably 92%, more preferably 95%, 98% sequence homogeny most preferably.

Perhaps, measure by the following method the sequence similarity degree between polynucleotides: multi-nucleotide hybrid, hybridization Condition can make the stable duplex of formation between homologous region, uses then the enzymic digestion of strand specific nucleic acid, measures subsequently to disappear Change the size of fragment. Measure order on definite molecular length of two nucleic acid or two peptide sequences when utilizing said method The row homogeny is at least about 70%-75%, preferred 80%-82%, and more preferably 85%-90%, more preferably 92%, more excellent Select 95%, most preferably 98% the time, then think their basic homologies. As used herein, basic homology also refers to specific DNA Or the identical sequence of peptide sequence. Utilize such as the Southern hybridization examination under the rigorous condition of concrete system regulation Test the dna sequence dna of identifying basic homology. Those skilled in the art can determine appropriate hybridization conditions. Referring to for example Sambrook etc., the same;" nucleic acid hybridization: practical approach " (Nucleic Acid Hybridization:A Practical Approach), B.D.Hames and S.J.Higgins compile, (1985) Oxford; The Washington D.C.; IRL Press).

Measure the selective cross of two nucleic acid fragments by following method. Sequence homogeny between two nucleic acid molecules Degree affects intermolecular hybridization efficiency and intensity. A part identical nucleic acid sequence can partly suppress and target at least The hybridization of the identical sequence of molecule. Utilize the cross experiment of being familiar with in following this area can measure identical sequence Hybridization suppress (such as Southern (DNA) marking, Northern (RNA) marking, solution hybridization etc., referring to Sambrook etc., " molecular cloning: laboratory manual " (Molecular Cloning; A Laboratory Manual), Second edition, (1989) Cold Spring Harbor, New York). This type of experiment can be carried out under in various degree selective, As utilizing from low to high different preciseness. If use low rigorous condition, can utilize lack in addition partial sequence identical The property second probe (for example being less than 30% probe with target molecule sequence homogeny) estimate and not have non-specific binding, when When lacking the non-specific binding event, second probe can not hybridized with target molecule.

When the detection system utilized based on hybridization, select the probe nucleic acid with the reference nucleic acid sequence complementation, and select Appropriate condition makes probe and the hybridization of reference sequences sequence selective or the formation bimolecular that interosculates. Can be medium rigorous Generally can hybridize under the following conditions with the nucleic acid molecules of reference sequences selective cross under the condition, this condition can Detect at least length and be 10-14 nucleotides and at least about the 70% sequence target nucleus identical with selected nucleic acid probe sequence Acid sequence. Rigorous hybridization conditions generally can detect with the sequence homogeny of selected nucleic acid probe sequence greater than approximately The length of 90-95% is at least the target nucleic acid sequence of about 10-14 nucleotides. Probe and ginseng sequence have specific growing up During the sequence homogeny of degree, the condition that is conducive to the hybridization of probe/reference sequences can be determined by technology known in the art (referring to For example" nucleic acid hybridization: practical approach " (Nucleic Acid Hybridization:A Practical Approach), B.D.Hames and S.J.Higgins compile, (1985) Oxford; The Washington D.C.; IRL Press).

Hybridization conditions is known for those skilled in the art. The hybridization preciseness refers to that hybridization conditions is unfavorable for comprising the mispairing nucleosides The degree that the hybridization son of acid forms, higher preciseness is relevant with lower tolerance to mismatch hybridization. Impact is assorted Hand over the factor of preciseness to know for those skilled in the art, include but not limited to: temperature, pH, ionic strength, assorted Hand over reaction time and organic solvent such as the concentration of for example formamide and dimethyl sulfoxide (DMSO). As is known to the person skilled in the art, Higher temperature, lower ionic strength and lower solvent strength will increase the hybridization preciseness.

As for the preciseness condition of hybridization, well known adopt many equivalent conditions by change (for example) following because of Usually set up specific preciseness: the base composition of the length of sequence and characteristic, various sequences, salinity and other There are or do not exist blocking agent (such as sulfuric acid dextran and polyethylene glycol), hybridization in hybridization solution component, the hybridization solution Reaction temperature and time parameter and wash conditions. According to this area standard method (referring to for example Sambrook, etc.," molecular cloning: laboratory manual " (Molecular Clining:A Laboratory Manual), second edition, (1989) Cold Spring Harbor, New York) select the concrete setting of hybridization conditions.

" combination " refers to sequence-specific, the noncovalent interaction between the macromole (as protein and nucleic acid).The all components of combination differs, and to establish a capital need be sequence-specific (as contacting with phosphoric acid residue in the dna backbone), and it is sequence-specific on the whole that pharmacy interacts.This interactional general features is dissociation constant (K _d) be 10 ^-6M ^-1Or it is lower." affinity " refers to bond strength: the binding affinity of increase and low K _dBe associated.

" conjugated protein " is the protein that can non-covalently be incorporated into another molecule.Conjugated protein can be in conjunction with (for example) dna molecular (DNA is conjugated protein), RNA molecule (rna binding protein) and/or protein molecule (protein bound albumen).Under the proteic situation of protein bound, but it self can be incorporated into one or more molecules of different proteins in conjunction with (forming homodimer, homotrimer etc.) and/or it.Conjugated protein combination activity with more than one types.For example have DNA combination, RNA combination and the active zinc finger protein of protein bound.

" zinc finger dna is conjugated protein " (or zinc refers in conjunction with the territory) is to refer in conjunction with the protein of DNA or than the domain of large protein by one or more zinc in the sequence-specific mode, and zinc refers to it is amino acid sequence region by in the binding structural domain of zinc ion coordination rock-steady structure.Conjugated protein zinc finger protein or the ZFP of usually abbreviating as of term zinc finger dna.

Zinc refers to can be through " engineered " with the predetermined nucleotide sequence of combination in conjunction with the territory.The non-limitative example of the method for engineered zinc finger protein is design and selects.

The zinc finger protein of design is a non-existent protein under the natural situation, and its design/composition is mainly from reasonable standard.The reasonable standard of design comprises and use to replace rule and computerized algorithm, and this algorithm is used for processing the information of the database of information that stores existing ZFP design and binding data.Referring to for example United States Patent (USP) 6,140,081; 6,453,242 and 6,534,261; Also referring to WO98/53058; WO98/53059; WO98/53060; WO02/016536 and WO03/016496.

" selecting " zinc finger protein is undiscovered protein under the natural situation, and it is produced mainly from empirical process, presents, acts on trap or hybridization selection as phage.Referring to for example US 5,789,538; US 5,925, and 523; US6,007,988; US 6,013, and 453; US 6,200, and 759; WO95/19431; WO96/06166; WO98/53057; And WO98/54311; WO00/27878; WO01/60970; WO01/88197 and WO02/099084.

Therefore, " engineered zinc finger protein " refers to contain the protein that one or more zinc refer to, it can be incorporated into predetermined nucleotide sequence in the sequence-specific mode through making up.Usually, engineered zinc finger protein is that the protein of non-natural generation and/or the zinc that contains the natural generation of arranging and/or making up in non-natural generation ground refer to.Binding specificity that engineered zinc refers to and the method that makes up engineered zinc finger protein include but not limited to: appropriate design, randomization/selection technology, polysome are selected, cis presents (cis-display), single crosses and two-hybrid system and select from the randomization library that the zinc of engineered and natural generation refers to.Referring to for example United States Patent (USP) 5,789,538; 6,007,988; 6,013,453; 6,140,466; 6,242,568; 6,410,248; 6,453,242; 6,479,626; 6,503,717; 6,534,261; 6,706,470; 6,733,970 and 6,746,838; U.S. Patent Application Publication 2003/0044957; 2003/0068675; 2003/0104526; 2003/0108880; 2003/0166141 and 2004/0091991; With the open WO 98/53057 of PCT; WO 98/53058; WO 98/53059; WO 98/53060; WO 01/53480; WO 01/88197; With WO 02/77227, by being used for all purposes with reference to including its content in this paper in full.Term " engineered zinc finger protein " does not refer to zinc finger protein that clone, natural generation.

" chromatin " is the nucleoprotein structure that comprises cellular genome.Cyto-chromatin comprises nucleic acid (mainly being DNA) and protein, and protein comprises histone and nonhistone chromosomal protein.Most of eukaryotic chromatins exist with the form of nucleosome, and wherein nucleosome core comprises the DNA of about 150 base pairs, and comprise each a pair of octamer of histone H2A, H2B, H3 and H4 and link to each other; Linker DNA (because of the organism different length variable) between nucleosome core, extend.The histone h1 molecule links to each other with linker DNA usually.For the object of the invention, term " chromatin " is intended to comprise all types cell, comprises protokaryon and eukaryotic nucleoprotein.Cyto-chromatin comprises chromosome and episome chromatin.

" chromosome " is all or part of the chromatin complex that comprises cellular genome.The feature of cellular genome usually is its caryogram, and it is all chromosomal set that comprise this cellular genome.Cellular genome can comprise one or more chromosome.

" episome " is replicating nucleic acid, nucleoprotein complex or other structure that comprises the nucleic acid that is not a cell chromosome caryogram part.Episomal example comprises plasmid and some viral genome.

" target site " or " target sequence " is certain nucleotide sequence, if it has determined to exist the bonded condition that is enough to, binding molecule (as conjugated protein) can bonded nucleic acid moiety.For example sequence 5 '-GAATTC-3 ' is the target site of Eco RI restriction endonuclease.

" can and district " be the bonded cyto-chromatin of exogenous molecule that the target site that exists in the nucleic acid of site can be identified target site.If do not wish to be subject to any concrete theory, should believe that can and distinguish is the zone that is not packaged in the nucleosomal structure.Usually can be by the unique texture that the sensitivity Detection of chemical probe and enzyme probe such as ribozyme can and be distinguished.

" exogenous " molecule is usually not to be present in the cell but the molecule in available one or more heredity, biochemistry or other method introducing cell.Concrete stage of development and environmental condition according to cell are determined " being present in the cell usually ".Therefore, the molecule that for example only exists during the fetal development of muscle is the exogenous molecule of adult's muscle cell.Similarly, the molecule of heat-inducible is the exogenous molecule of non-heat shock cell.Exogenous molecule can comprise the malfunction type that function type or normally functioning endogenous molecule are arranged of the endogenous molecule of (for example) malfunction.

Exogenous molecule can be micromolecule or macromole etc., the molecule that produces of micromolecule such as combinational chemistry wherein, the modified derivative of macromole such as protein, nucleic acid, sugar, lipid, glycoprotein, lipoprotein, polysaccharide, above-mentioned molecule, or comprise the complex of one or more above-mentioned molecules.Nucleic acid comprises DNA and RNA, can be strand or two strands; Can be straight chain, side chain or annular; Can be any length.Nucleic acid comprises the nucleic acid that can form duplex, and the nucleic acid that forms triplex.Referring to for example U.S. Patent number 5,176,996 and 5,422,251.Protein includes but not limited to: DNA is conjugated protein, transcription factor, the chromatin remodeling factor, methylate DNA are conjugated protein, polymerase, methylase, demethylase, acetyltransferase, deacetylase, kinases, phosphatase, intergrase, recombinase, ligase, topoisomerase, gyrase and unwindase.

Exogenous molecule can be the molecule identical with the endogenous molecule type, as exogenous protein or nucleic acid.For example, exogenous nucleic acid can comprise infectious virus genome, the plasmid of introducing cell or episome or the common chromosome that is not present in the cell.The method of exogenous molecule being introduced cell is well known by persons skilled in the art, include but not limited to: the transfer of the transfer (be liposome, comprise neutrality and cation lipid) of lipid mediation, electroporation, direct injection, cell fusion, particle bombardment, coprecipitation of calcium phosphate, the mediation of DEAE-dextran and the transfer of viral vector mediation.

On the contrary, " endogenous " molecule is the molecule that exists usually under concrete environmental condition, in the concrete cell of concrete stage of development.For example, endogenous nucleic acid can comprise chromosome, the genome of mitochondrion, chloroplast or other organelle, or the episome nucleic acid of natural generation.Other endogenous molecule can comprise protein, for example transcription factor and enzyme.

" fusion " molecule is that two or more subunit molecules connect, the molecule that preferably covalently connects.The subunit molecule can be the molecule of identical chemical type, perhaps can be the molecule of different chemical type.The example of first type fusion molecule includes but not limited to: fusion rotein (for example ZFP DNA is in conjunction with the fusions between territory and the transcriptional regulatory territory) and integrative nucleic acid (nucleic acid of the above-mentioned fusion rotein of for example encoding).The example of second type fusion molecule includes but not limited to: form the nucleic acid of triplex and the fusions between the fusions between the polypeptide and minor groove binders and the nucleic acid.

Can cause protein (as fusion rotein) to be expressed in cell in the cell by protein delivery is delivered in cell or by the polynucleotide with coded protein, wherein polynucleotide be transcribed, and transcript is translated, to produce protein.Also can adopt the RNA molecule is delivered in the cell, in cell, translate subsequently, with marking protein in cell.Marking protein can comprise that also trans-splicing, polypeptide cutting are connected with polypeptide in cell.Is known in the art with polynucleotide and polypeptide delivery to the method in the cell, and exemplary method has been listed in other place of the disclosure.

For disclosure purpose, " gene " comprises the DNA zone (as follows) of encoding gene product, and all DNA zones of the generation of regulator gene product, and territory coding and/or transcription sequence are adjacent no matter this kind regulated sequence.Therefore, gene includes but not limited to: sequence such as ribosome binding site and internal ribosome entry site, enhancer, silencer, insulator, boundary element, origin of replication, substrate attachment site and locus control region are regulated in promoter sequence, terminator, translation.

" gene expression " refers to change the contained information of gene into gene outcome.Gene outcome can be the direct transcription product (as the RNA of mRNA, tRNA, rRNA, antisense dsRNA, ribozyme, structure RNA or any other type) of gene, the transcript of processing such as the protein of siRNA or the generation of mRNA antisense.Gene outcome also comprises by certain methods as adding medicated cap, polyadenylation, methylate and edit the RNA of modification, and methylate by (for example), acetylation, phosphorylation, ubiquitinization, ADP-ribosylation, myristylation and glycosylation modified protein.

Gene expression " adjusting " refers to that gene activity changes.Express and regulate and can include but not limited to: gene activation and gene are prevented.

" eucaryon " cell includes but not limited to: fungal cell's (as yeast), plant cell, zooblast, mammalian cell and people's cell.

" area-of-interest " is any zone of cyto-chromatin, and for example gene or gene are interior or the non-coding sequence adjacent with gene, wherein need this zone in conjunction with exogenous molecule.In conjunction with can being for (for example) transcriptional regulatory.Area-of-interest can be present in (for example) chromosome, episome, organelle gene group (as mitochondrion, chloroplast) or the infectious virus genome.Area-of-interest can be in the coding region of gene, in noncoding region of transcribing such as targeting sequencing, tailer sequence or the intron, perhaps in the nontranscribed domain in upstream of coding region or downstream.Area-of-interest may be as little to single nucleotide pair or reaches 2,000 nucleotide pairs, or any integer nucleotide pair.

Term " operability connection " is used interchangeably with " operability is connected " (or " can be operatively connected "), refer to two or more parts (as sequential element) arranged side by side, wherein the arrangement of parts can make these parts function of bringing into normal play, and the effect that at least one parts mediation is applied at least one other parts.For example, if transcriptional regulatory sequences according to the existence of one or more transcription regulaton factors or there is not the transcriptional level of having controlled coded sequence, this transcriptional regulatory sequences such as promoter operability are connected in this coded sequence so.Transcriptional regulatory sequences is connected with coded sequence cis operability usually, but not necessarily directly is adjacent.For example, enhancer is the transcriptional regulatory sequences that operability is connected in coded sequence, even they do not adjoin.

When mentioning fused polypeptide, carry out identical functions when not being connected like this with it when term " operability connection " can refer to that each parts is connected with other parts.For example, when mentioning ZFP DNA and being blended in the fused polypeptide in transcriptional regulatory territory in conjunction with the territory, if ZFP DNA-can be in conjunction with its target site and/or its binding site in conjunction with the territory part in fused polypeptide, can regulate (as activation or prevent) and transcribe and regulate the territory, ZFP DNA links to each other in conjunction with territory and adjusting territory operability so.

" functional equivalent " of protein, polypeptide or nucleic acid or " function fragment " they are that sequence is different with full length protein, polypeptide or nucleic acid, but protein, polypeptide or the nucleic acid of reservation and full length protein, polypeptide or nucleic acid identical functions.The residue quantity of function fragment can be more than the natural molecule of correspondence, less or identical, and/or function fragment can contain one or more aminoacid or nucleotide replaces.Measure nucleic acid function (as encoding function, with the ability of another nucleic acid hybridization) method be well known in the art.Similarly, the method for measuring protein function is known.For example, can be by the DNA combined function of the combination of (for example) filter membrane, electrophoretic mobility change or immunoprecipitation test determination polypeptide.Can measure the DNA cutting by gel electrophoresis.Referring to Ausubel etc., the same.Can pass through the ability of (for example) co-immunoprecipitation, double cross mensuration or complementary (heredity and biochemical) mensuration protein and another kind of protein interaction.Referring to (1989) Nature 340:245-246 such as for example Fields; U.S. Patent number 5,585,245 and PCT WO 98/44350.

Engineered zinc finger protein and target sequence

Herein disclosed is the compositions and the method that are used for transcriptional regulatory, this method can be used for (for example) increases RNA and/or proteinic output.They comprise the fusion rotein that comprises engineered zinc finger protein and functional domain (for example) transcription activating domain.Suitable functional domain is known in the art, includes but not limited to: transcription activating domain such as VP16, VP64 and p65.And, can there be one or more identical or different functional domains (as transcription activating domain) in the given fusion rotein.Referring to by with reference to the total U.S. Patent Application Publication No. 2002/0160940 of including this paper in, the exemplary functionality territory is wherein disclosed.

In some embodiments, engineered zinc finger protein makes it in conjunction with the sequence that comprises target sequence GCTGTGGAA (SEQ ID NO:1).This sequence is present in the promoter SR α promoter (Takebe etc., the same) that is generally used for protein production, but one or more SEQ ID NO:1 copy can be inserted in any promoteres known in the art (as the CMV promoter) or adjacent with promoter.

Can refer to that zinc finger protein SBS2392/00 has following aminoacid sequence in conjunction with exemplary three of SEQ ID NO:1 through engineered:

KKKQHICHIQGCGKVYG QRSNLVRHLRWHTGERPFMCTWSYCGKRFT RSD ALSRHKRTHTGEKKFACPECPKRFM QSSDLRRHIKTHQNK(SEQ?ID?NO：2)。

The amino acid residue of underscore to+6 (a spiral part that refers in zinc begins the place), and is called as " cog region " corresponding to residue-1 among the SEQ ID NO:2, because one or more bonded sequence-specifics of nucleic acid that participated in these residues.Therefore, the protein that comprises three identical cog regions in homopolypeptide main chain sequence not is considered to be designated as the proteinic equivalent of SEQ ID NO:2, because they have identical DNA binding specificity.

Therefore, in some embodiments, these three cog regions (shown in the underscore of above-mentioned SEQ ID NO:2) can be put into any zinc and refer to that main chain is (referring to for example United States Patent (USP) 6,453,242 and 6,534,261), the protein that obtains can be used for regulating transcribes, and (for example) is to improve protein output.Therefore, the engineered zinc finger protein with following sequence can be used for described method:

C-X _2-4-C-X ₅-QRSNLVR-H-X _3-5-H-X ₇-C-X _2-4-C-X ₅-RSDALSR-H-X _3-5-H-X ₇-C-X _2-4-C-X ₅-QSSDLRR-H-X _3-5-H(SEQ?ID?NO：3)。

In cog region, residue-1 ,+3 and+6 main protein-nucleotide of being responsible for contact.Therefore, the non-limitative example of other equivalent comprises and contains the protein that three zinc refer to that wherein first zinc refers to contain the Q residue at-1, contains the N residue at+3, contains R residue (QXXNXXR, SEQ ID NO:4) at+6; Second zinc refers to contain the R residue at-1, contains the A residue at+3, contains R residue (RXXAXXR, SEQID NO:5) at+6; The 3rd zinc refers to contain the Q residue at-1, contains the D residue at+3, contains R residue (QXXDXXR, SEQ ID NO:6) at+6.Other equivalent comprises any ZFP that is incorporated into the sequence that contains target sequence GCTGTGGAA (SEQ ID NO:1).

Have following aminoacid sequence through engineered other exemplary three finger zinc finger protein SBS2392/10 that are incorporated into target sequence GCTGTGGAA (SEQ ID NO:1):

KKKQHICHIQGCGKVYG QSSNLARHLRWHTGERPFMCTWSYCGKRFT RSD ALTRHKRTHTGEKKFACPECPKRFM QSCDLTRHIKTHQNK(SEQ?ID?NO：7)。The amino acid residue of underscore to+6 (the α spiral part that refers in zinc begins the place), and is called as " cog region " corresponding to residue-1 among the SEQID NO:7, because one or more bonded sequence-specifics of nucleic acid that participated in these residues.Therefore, the protein that comprises three identical cog regions in homopolypeptide main chain sequence not is considered to the equivalent of SEQ ID NO:7, because they have identical DNA binding specificity.

Therefore, in some embodiments, these three cog regions (shown in the underscore of above-mentioned SEQ ID NO:7) can be put into any zinc and refer to that main chain is (referring to for example United States Patent (USP) 6,453,242 and 6,534,261), the protein that obtains can be used for regulating transcribes, and (for example) is to increase protein output.Therefore, the engineered zinc finger protein with following sequence can be used for described method:

C-X _2-4-C-X ₅-QSSNLAR-H-X _3-5-H-X ₇-C-X _2-4-C-X ₅-RSDALTR-H-X _3-5-H-X ₇-C-X _2-4-C-X ₅-QSCDLTR-H-X _3-5-H(SEQ?ID?NO：8)。

In cog region, residue-1 ,+3 and+6 main protein-nucleotide of being responsible for contact.Therefore, the non-limitative example of other equivalent comprises and contains the protein that three zinc refer to that wherein first zinc refers to contain the Q residue at-1, contains the N residue at+3, contains R residue (QXXNXXR, SEQ ID NO:4) at+6; Second zinc refers to contain the R residue at-1, contains the A residue at+3, contains R residue (RXXAXXR, SEQID NO:5) at+6; The 3rd zinc refers to contain the Q residue at-1, contains the D residue at+3, contains R residue (QXXDXXR, SEQ ID NO:6) at+6.Therefore, (for example) protein of comprising SEQ ID NO:25 is considered to be used for the equivalent of described method.

C-X _2-4-C-X ₅-QXXNXXR-H-X _3-5-H-X ₇-C-X _2-4-C-X ₅-RXXAXXR-H-X _3-5-H-X ₇-C-X _{2 -4}-C-X ₅-QXXDXXR-H-X _3-5-H(SEQ?ID?NO：25)

Other equivalent comprises any ZFP that is incorporated into the sequence that comprises target sequence GCTGTGGAA (SEQ ID NO:1).

Described zinc refer to cog region-1 ,+3 and+correspondence between the aminoacid at 6 contact residues places and the nucleotide of target site.Referring to for example U.S. Patent number 6,007,988; 6,013,453 and 6,746,838; And the open WO 96/06166 of PCT; WO 98/53058; WO 98/53059 and WO 98/53060.Therefore, also the equivalent of Kao Lving is three finger zinc finger proteins, and wherein first refers to contain Q at-1 place; Contain N at+3 places; Contain R, K, S or T at+6 places; Second refers to contain R at-1 place; Contain A, S or V at+3 places, contain R, K, S or T at+6 places; The 3rd refers to contain N, Q, H or T at-1 place; Contain S, D, E, L, T or V at+3 places, contain R, K, S or T at+6 places.

Fusion rotein and coded polynucleotide

As mentioned above, engineered Zinc-finger DNA binding domain as herein described can comprise a part of fusion rotein, and wherein fusion rotein also contains one or more functional domains (as the transcriptional regulatory territory), nuclear localization sequence, epi-position label etc.For example, comprise nuclear localization sequence (NLS), zinc refers to be called 2392/00 in conjunction with the aminoacid sequence of the fusion rotein of territory (ZFP), VP16 transcription activating domain (VP16) and FLAG epi-position label (FLAG), sees Fig. 2 (SEQ ID NO:10).This proteic nucleotide sequence of encoding is seen Fig. 3 (SEQ ID NO:11).

Other exemplary proteins (2392/10) has aminoacid sequence shown in Figure 4 (SEQ ID NO:12).The 2392/10 proteic polynucleotide sequence (SEQ ID NO:13) of encoding is seen Fig. 5.

Functional domain

Any DNA in conjunction with the territory all can be randomly with help (as) DNA processing (as the DNA cutting) or gene expression one or more " functional domains " or " adjusting territory " regulated links to each other.Can covalently or non-covalently be connected in one or more adjustings territory in conjunction with the territory, perhaps two or more adjustings territory, these two or more domains are two copies of same domain, or two different structure territories.Regulate the territory can (as) be covalently attached in conjunction with the territory by the aminoacid joint, become the part of fusion rotein.DNA also can regulate the territory (referring to for example O ' Shea by non-covalent dimerization domain such as conjugated protein being connected in of leucine zipper, stat protein N-terminal domain or FK506 in conjunction with the territory, Science 254:539 (1991), Barahmand-Pour etc., Curr.Top.Microbiol.Immunol.211:121-128 (1996); Klemm etc., Annu.Rev.Immunol.16:569-592 (1998); Klemm etc., Annu.Rev.Immunol.16:569-592 (1998); Ho etc., Nature 382:822-826 (1996); With Pomeranz etc., Biochem.37:965 (1998)).Regulate the territory and can pass through any correct position, comprise that C-terminal or the N-terminal in conjunction with the territory is connected in conjunction with the territory.

Common adjusting territory for example comprises: the effector domain of transcription factor (activator, repressor, co-activation thing, common repressor), reticent thing, nuclear hormone receptor, oncogene transcription factor (as myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family member etc.); DNA repairase and correlation factor thereof and trim; DNA resets enzyme and correlation factor and trim; Chromatin associated protein and trim thereof (as kinases, acetyltransferase and deacetylase); With dna modification enzyme (as transmethylase, topoisomerase, unwindase, ligase, kinases, phosphatase, polymerase, Cobra venom endonuclease, intergrase) and correlation factor and trim.

The transcription factor polypeptide that can therefrom obtain to regulate the territory comprises and participates in regulating and the polypeptide of basal transcription.These polypeptide comprise transcription factor, their effector domain, co-activation thing, reticent thing, nuclear hormone receptor, and (referring to for example Goodrich etc., Cell 84:825-30 (1996) is about the protein that participates in transcribing and the summary of nucleic acid elements; Usually see Barnes and Adcock about the summary of transcription factor, Clin.Exp.Allergy 25 supplementary issue 2:46-9 (1995) and Roeder, Methods Enzymol.273:165-71 (1996)).The known data base who is specifically designed to transcription factor (referring to for example Science 269:630 (1995) and TRANSFAC).At (for example) Rosen etc., J.Med.Chem.38:4855-74 has described the nuclear hormone receptor transcription factor in (1995).The summary of the C/EBP family of transcription factor is seen Wedel etc., Immunobiology 193:171-85 (1995).By the co-activation thing of nuclear hormone receptor mediation transcriptional regulatory and altogether the summary of repressor referring to for example Meier, Eur.J.Endocrinol.134 (2): 158-9 (1996); Kaiser etc., Trends Biochem.Sci.21:342-5 (1996); With Utley etc., Nature 394:498-502 (1998)).The description that participates in the GATA transcription factor that hemopoietic regulates is referring to for example Simon, Nat.Genet.11:9-11 (1995); Weiss etc., Exp.Hematol.23:99-107.Goodrich and Tjian, Curr.Opin.Cell Biol.6:403-9 (1994) and Hurley have described TATA frame conjugated protein (TBP) and relevant TAF polypeptide (comprising TAF30, TAF55, TAF80, TAF110, TAF150 and TAF250) thereof among the Curr.Opin.Struct.Biol.6:69-75 (1996).The summary of the STAT family of transcription factor is referring to for example Barahmand-Pour etc., Curr.Top.Microbiol.Immunol.211:121-8 (1996).The summary of the transcription factor of involved in diseases is seen Aso etc., J.Clin.Invest.97:1561-9 (1996).

In one embodiment, prevent domain and/or KRAB to prevent domain to be used as transcription repressor the proteic KOX of people KOX-1.Thiesen etc., New Biologist 2:363-374 (1990); Margolin etc., PNAS91:4509-4513 (1994); Pengue etc., Nucl.Acids Res.22:2908-2914 (1994); Witzgall etc., PNAS 91:4514-4518 (1994).In another embodiment, the common repressor KAP-1 of KRAB uses with KRAB or KOX.Friedman etc., Genes Dev.10:2067-2078 (1996).Perhaps, KAP-1 can be used alone as functional domain.As other preferred transcription factor of transcription repressor and transcription factor domain comprise MAD (referring to for example Sommer etc., J.Biol.Chem.273:6632-6642 (1998); Gupta etc., Oncogene 16:1149-1159 (1998); Queva etc., Oncogene 16:967-977 (1998); Larsson etc., Oncogene 15:737-748 (1997); Laherty etc., Cell 89:349-356 (1997); With Cultraro etc., Mol Cell.Biol.17:2353-2359 (19977)); FKHR (the forkhead of rhabdomyosarcoma (rhapdosarcoma) gene; Ginsberg etc., Cancer Res.15:3542-3546 (1998); Epstein etc., Mol.Cell.Biol.18:4118-4130 (1998)); EGR-1 (early growth response gene product-1; Yan etc., PNAS 95:8298-8303 (1998); With Liu etc., Cancer Gene Ther.5:3-28 (1998)); The ets2 repressor factor is prevented domain (ERD; Sgouras etc., EMBO are ((19095)) J.14:4781-4793; With MAD smSIN3 action scope (SID; Ayer etc., Mol.Cell.Biol.16:5772-5781 (1996)).

In one embodiment, HSV VP16 activation domain is as transcriptional activator (referring to for example Hagmann etc., J.Virol.71:5952-5962 (1997)).Can comprise nuclear hormone receptor (referring to for example Torchia etc., Curr.Opin.Cell.Biol.10:373-383 (1998)) by its other transcription factor that obtains activation domain; The p65 subunit of nuclear factor κ B (Bitko and Barik, J.Virol.72:5610-5618 (1998) and Doyle and Hunt, Neuroreport 8:2937-2942 (1997)); And EGR-1 (early growth response gene product-1; Yan etc., PNAS 95:8298-8303 (1998); With Liu etc., Cancer Gene Ther.5:3-28 (1998)).Other synthetic activation domain is VP64 activation domain (Seipel etc., EMBO are (1996) J.11:4961-4968).

Other protein that kinases, phosphatase, methylase, demethyl enzyme, acetyltransferase, deacetylase and modification participate in the polypeptide of Gene regulation also can be used as the adjusting territory.These instrumentalities have usually participated in (for example) hormone-mediated initial or termination of transcribing.The kinase whose summary that participates in transcriptional regulatory is seen Davis, Mol.Reprod.Dev.42:459-67 (1995), Jackson etc., Adv.Second Messenger Phosphoprotein Res.28:279-86 (1993) and Boulikas, Crit.Rev.Eukaryot.Gene Expr.5:1-77 (1995), and the summary of phosphatase is referring to for example Schonthal and Semin, Cancer Biol.6:239-48 (1995).Wang, Trends Biochem.Sci.19:373-6 has described the nuclear tyrosine kinase in (1994).

As mentioned above, useful domain also can be available from gene outcome (as myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family member) and correlation factor and the trim of oncogene.Cooper for example, " oncogene " (Oncogenes), the 2nd edition, The Jones ﹠amp; Bartlett Series in Biology, Boston, Massachusetts, Jones and BartlettPublishers have described oncogene in 1995.The summary of ets transcription factor is seen Waslylk etc., Eur.J.Biochem.211:7-18 (1993) and Crepieux etc., Crit.Rev.Oncog.5:615-38 (1994).The summary of Myc oncogene is referring to for example Ryan etc., Biochem.J.314:713-21 (1996).The description of jun and fos transcription factor is referring to for example " Fos of transcription factor and Jun family " (The Fos andJun Families of Transcription factors), and Angel and Herrlich compile (1994).The summary of max oncogene is referring to Hurlin etc., Cold Spring Harb.Symp.Quant.Biol.59:109-16.The summary of myb gene family is referring to Kanei-Ishii etc., Curr.Top.Microbiol.Immunol.211:89-98 (1996).The summary of mos family is referring to Yew etc., Curr.Opin.Genet.Dev.3:19-25 (1993).

Regulating the territory also can be available from DNA repairase and correlation factor and trim.They comprise (for example) nuclease (inscribe and circumscribed), recombinase, unwindase, intergrase, polymerase and single-stranded DNA binding protein (SSB).The summary of DNA repair system is referring to for example Vos, Curr.Opin.Cell Biol.4:385-95 (1992); Sancar, Ann.Rev.Genet.29:69-105 (1995); Lehmann, Genet.Eng.17:1-19 (1995); And Wood, Ann.Rev.Biochem.65:135-67 (1996).DNA reset enzyme and correlation factor and trim thereof also can be used as regulate the territory (referring to for example Gangloff etc., Experientia 50:261-9 (1994); Sadowski, FASEB be (1993) J.7:760-7).

Similarly, regulating the territory can be available from dna modification enzyme (as dnmt rna, topoisomerase, unwindase, ligase, kinases, phosphatase, polymerase) and correlation factor and trim.The summary of unwindase is referring to Matson etc., Bioessays, 16:13-22 (1994); Cheng, Curr.Opin.Struct.Biol.5:4-10 has described transmethylase in (1995).Chromatin associated protein and trim thereof (as kinases, acetyltransferase and deacetylase), (Wolffe, Science 272:371-2 (1996)) also can be used as functional domain as histone deacetylase.In one embodiment, regulate the territory and be dnmt rna as transcription repressor (referring to for example Van den Wyngaert etc., FEBS Lett.426:283-289 (1998); Flynn etc., J.Mol.Biol.279:101-116 (1998); Okano etc., Nucleic Acids Res.26:2536-2540 (1998); With Zardo and Caiafa, J.Biol.Chem.273:16517-16520 (1998)).In another embodiment, Cobra venom endonuclease such as Fok1 provide functional domain with catalysis targeting DNA cutting, and this helps following method, for example transcription repression and homologous recombination.Referring to for example United States Patent (USP) 5,436,150; 5,792,640 and 6,265,196; U.S. Patent Application Publication No. 2003/0232410 and WO03/87341.

Control chromatin and dna structure, mobile and the localized factor and correlation factor and trim; The functional domain that also can be used for obtaining to be used to make up chimeric protein or fusion molecule derived from the factor of microorganism (as prokaryote, eukaryote and virus) and the factor relevant with it or that modify them.In one embodiment, recombinase and intergrase are as regulating the territory.In another embodiment, the histone acetyl based transferase as transcription activating domain (referring to for example Jin and Scotto, Mol.Cell.Biol.18:4377-4384 (1998); Wolffe, Science 272:371-372 (1996); Taunton etc., Science 272:408-411 (1996); With Hassig etc., PNAS 95:3519-3524 (1998)).In another embodiment, histone deacetylase as the transcription repression domain (referring to for example Jin and Scotto, Mol.Cell.Biol.18:4377-4384 (1998); Syntichaki and Thireos, J.Biol.Chem.273:24414-24419 (1998); Sakaguchi etc., Genes Dev.12:2831-2841 (1998); With Martinez etc., J.Biol.Chem.273:23781-23785 (1998)).

The another kind of suitable domain of preventing is methyl binding domain protein 2B (MBD-2B) (also referring among (1999) Mamm Genome 10:906-912 such as Hendrich to the proteic description of MBD).The another kind of useful domain of preventing is the domain relevant with v-ErbA albumen.Referring to (1989) Nature 339:593-597 such as for example Damm; Evans (1989) Int.J.Cancer supplementary issue 4:26-28; Pain etc. (1990) New Biol.2:284-294; Sap etc. (1989) Nature 340:242-244; Zenke etc. (1988) Cell 52:107-119; With (1990) Cell 61:1035-1049 such as Zenke.Other exemplary domain of preventing includes but not limited to: and Thyroid Hormone Receptors (TR, as follows), SID, MBD1, MBD2, MBD3, MBD4, MBD sample albumen, DNMT family member (as DNMT1, DNMT3A, DNMT3B), Rb, MeCP1 and MeCP2.Referring to (1999) Cell 99:451-454 such as for example Bird; Tyler etc. (1999) Cell 99:443-446; Knoepfler etc. (1999) Cell 99:447-450; With (2000) Nature Genet.25:338-342 such as Robertson.Other exemplary domain of preventing includes but not limited to: ROM2 and AtHD2A.Referring to (1996) Plant Cell 8:305-321 such as for example Chern; With (2000) Plant such as Wu J.22:19-27.

Some member of nuclear hormone receptor (NHR) superfamily for example comprises that Thyroid Hormone Receptors (TR) and retinoic acid receptors (RAR) are the most effective known transcriptional at present.Zhang etc., Annu.Rev.Physiol.62:439-466 (2000) and Sucov etc., Mol Neurobiol 10 (2-3): 169-184 (1995).Under the situation that does not have its related part, these albumen are incorporated into regulator gene seat (as enhancer and promoter) the short section of interior DNA (as 12-17 base pair) with high specific and affinity, and realize the potent transcription repression to adjacent gene.The effectiveness of their regulating actions originates from and adopts two difference in functionality approach to drive gene silencing simultaneously: (i) by targeting altogether the complex of repressor N-CoR and histone deacetylase HDAC3 produce and prevent chromatinic location structure territory (Guenther etc., Genes Dev 14:1048-1057 (2000); Urnov etc., EMBO J 19:4074-4090 (2000); Li etc., EMBO J 19,4342-4350 (2000) and Underhill etc., J.Biol.Chem.275:40463-40470 (2000)) and (ii) may comprise the basic transcriptional machinery function of direct interference (Fondell etc., Genes Dev 7 (7B): 1400-1410 (1993) and Fondell etc., the chromatinic approach (Urnov etc., the same) that do not rely on of Mol CellBiol 16:281-287 (1996).

When the concentration very low (as nanomole) of its part, these receptors carry out conformational change, cause common repressor discharges, collection (recruit) is dissimilar accessory molecule (as the co-activation thing) and effective transcriptional activation.Collingwood etc., J.Mol.Endocrinol.23 (3): 255-275 (1999).

Can be responsible for transcribing the receptor protein part of control (as prevent and activate) from being responsible for the bonded part physical separation of DNA, be connected in other polypeptide, it can keep repertoire during in conjunction with the territory as other DNA.Therefore, nuclear hormone receptor can be transcribed control domain and be blended in DNA, so that rely on DNA to make the interested chromosomal region of transcripting regulating activity targeting (as a kind of gene) of this receptor in conjunction with the territory in conjunction with territory (as zinc finger protein).

And, can so that it loses the whole abilities (thereby having lost the ability that drives transcriptional activation) to hormone response, but keep the ability that realizes transcription repression by the structure of natural method or recombinant technique change TR and other nuclear hormone receptor.The example of this method is the transcriptional regulatory characteristic of cancer protein v-ErbA.V-ErbA albumen is that fowl erythroblastosis virus makes the immature erythrocyte precursor of chicken carry out leukemia to transform one of necessary two kinds of albumen.TR is erythropoietic main instrumentality (Beug etc., Biochim Biophys Acta 1288 (3): M35-47 (1996); Specifically, under its non-part connection status, it can inhibitory cell Cycle Arrest and the required gene of differentiation state.Therefore, thyroxin is given immature hemocytoblast and cause their quick differentiation.The v-ErbA cancer protein is the TR that extensively suddenlys change; These sudden changes comprise: (i) 12 amino terminal amino acids of disappearance; (ii) be blended in the gag cancer protein; (iii) DNA is in conjunction with the several point mutation in the territory, and with respect to its parent TR, these point mutation have changed this proteic DNA binding specificity and weakened it and the ability of retinoid X receptor allos dimerization; (iv) this protein ligands is in conjunction with a plurality of point mutation in the territory, and the ability in conjunction with thyroxin has effectively been eliminated in these sudden changes; (v) lack one section aminoacid of c-terminus, this section aminoacid is that transcriptional activation is necessary.Stunnenberg etc., BiochimBiophys Acta 1423 (1): F15-33 (1999).These results of mutation are, v-ErbA has kept the ability of the TR target gene that is incorporated into natural generation, and in conjunction with the time be effective transcription repressor (Urnov etc., the same; Sap etc., Nature 340:242-244 (1989); With Ciana etc., EMBO is (24) J.17: 7382-7394 (1999).Yet opposite with TR, v-ErbA is insensitive fully to thyroxin, has therefore still kept transcription repression in the presence of thyroxin or retinoid.

Therefore, in one aspect, with v-ErbA or its function fragment as preventing domain.In other embodiments, when not having part with TR or its functional domain as prevent domain and/or when having part (as 3,5,3 '-three iodo-L-thyronine or T3) as activation domain.Therefore, TR can be used as convertible functional domain (being difunctional domain); Its activity (activate or prevent) depends on whether there is part respectively.

Other exemplary domain of preventing is available from DAX albumen and its function fragment.Zazopoulos etc., Nature390:311-315 (1997).Specifically, proved the C-end portion of DAX-1, comprised that aminoacid 245-470 has the activity of preventing.Altincicek etc., J.Biol.Chem.275:7662-7667 (2000).Other exemplary domain of preventing is RBP1 albumen and its function fragment.Lai etc., Oncogene 18:2091-2100 (1999); Lai etc., Mol.Cell.Biol.19:6632-6641 (1999); Lai etc., Mol.Cell.Biol.21:2918-2932 (2001) and WO 01/04296.Total length RBP1 polypeptide contains 1257 aminoacid.The exemplary functionality fragment of RBP1 is the polypeptide that comprises the polypeptide of amino acid/11 114-1257 and comprise aminoacid 243-452.

The TIEG family member of transcription factor is contained three and is prevented domain, is called R1, R2 and R3.Preventing to small part of TIEG family protein realizes by collection mSIN3A histone deacetylase complex.Cook etc. (1999) J.Biol.Chem.274:29,500-29,504; Zhang etc. (2001) Mol.Cell.Biol.21:5041-5049.Any or all these prevent domain (or its function fragment) to prevent domain (or its function fragment) to be blended in DNA individually or with other in conjunction with the territory, to produce the exogenous molecule of preventing of targeting.

And, with the product of Human Cytomegloviru (HCMV) UL34 open reading frame as some HCMV gene, as US3 gene transcription repressor.LaPierre etc. (2001) J.Virol.75:6062-6069.Therefore, UL34 gene outcome or its function fragment can be used as and also comprise zinc and refer to parts in conjunction with the fused polypeptide in territory.The nucleic acid of this fusions of encoding also can be used for methods described herein and compositions.

The another exemplary domain of preventing is CDF-1 transcription factor and/or its function fragment.Referring to for example WO99/27092.

Ikaros protein family to small part has participated in the adjusting that lymphocyte is grown by transcription repression.Therefore, Ikaros family member (as Ikaros, Aiolos) or its function fragment can be used as and prevent domain.Referring to (2001) EMBO such as for example Sabbattini J.20:2812-2822.

Yeast Ash1p albumen comprises the transcription repression domain.Maxon etc. (2001) Proc.Natl.Acad.Sci.USA98:1495-1500.Therefore, the congener of Ash1p albumen, its function fragment and Ash1p, as the congener that is found in (for example) vertebrates, mammal and the plant cell can be used as the domain of preventing that is used for methods described herein and compositions.

Other exemplary domain of preventing comprises derived from the domain of preventing of organizing material down: (HDAC is as I class HDAC for histone deacetylase, II class HDAC, the SIR-2 congener), the HDAC-interaction protein is (as SIN3, SAP30, SAP15, NCoR, SMRT, RB, p107, p130, RBAP46/48, MTA, Mi-2, Brg1, Brm), DNA-cytosine transmethylase is (as Dnmt1, Dnmt3a, Dnmt3b), in conjunction with the protein of methylate DNA (as MBD1, MBD2, MBD3, MBD4, MeCP2, DMAP1), protein methyltranferase is (as lysine and arginine methylase, SuVar congener such as Suv39H1), polycomb type repressor is (as Bmi-1, eed1, RING1, RYBP, E2F6, Mel18, YY1 and CtBP), virus receptor is (as adenovirus E 1 b 55K albumen, cytomegalovirus UL34 albumen, viral oncogene such as v-erbA), hormone receptor is (as Dax-1, estrogen receptor, Thyroid Hormone Receptors) and with the zinc finger protein of natural generation (as WT1, KAP1) the relevant domain of preventing.Other exemplary member and congener HPH1, HPH2, HPC2, NC2, groucho, Eve, tramtrak, mHP1, SIP1, ZEB1, ZEB2 and Enx1/Ezh2 that prevents domain to comprise the polycomb complex.Under all these situations, full length protein or function fragment all can be used as the domain of preventing that refers to combine the territory fusion with zinc.And, above-mentioned proteinic any congener, and with the protein (or its function fragment) of any above-mentioned protein interaction, also can be used as and prevent domain.

Other prevents domain and exemplary functionality fragment as follows.Hesl is people's congener of fruit bat (Drosophila) hairy gene outcome, comprises the function fragment that contains aminoacid 910-1014.Specifically, WRPW (trp-arg-pro-trp) motif can be used as and prevents domain.Fisher etc. (1996) Mol.Cell.Biol.16:2670-2677.

TLE1, TLE2 and TLE3 albumen are people's congeners of fruit bat groucho gene outcome.Have that to prevent active these proteic function fragments are amino acid/11s-400.Fisher etc., the same.

Tbx3 albumen has the functional domain of preventing of aminoacid 524-721.He etc. (1999) Proc.Natl.Acad.Sci.USA 96:10,212-10,217.The Tbx2 gene outcome has participated in preventing of p14/p16 gene, and it comprises the zone of preventing the homologous aminoacid 504-702 of domain with Tbx3; Therefore, Tbx2 and/or this function fragment can be used as and prevent domain.Carreira etc. (1998) Mol.Cell.Biol.18:5,099-5,108.

People Ezh2 albumen is the congener of fruit bat zeste enhancer, and can collect eed1 polycomb type repressor.The Ezh2 albumen zone that comprises amino acid/11-193 can and be prevented and transcribe with the eed1 interaction; Therefore, Ezh2 and/or this function fragment can be used as and prevent domain.Denisenko etc. (1998) Mol.Cell.Biol.18:5634-5642.

RYBP albumen is the common repressor with polycomb complex member and YY1 transcription factor interaction.The RYBP zone that comprises aminoacid 42-208 is accredited as the functional domain of preventing.Garcia etc. (1999) EMBO J.18:3404-3418.

RING finger protein RING1A is the member of two kinds of different vertebrates polycomb type complex, and it comprises a plurality of binding sites of the various assemblies of polycomb complex, and has the transcription repression activity.Therefore, RING1A or its function fragment can be used as and prevent domain.Satjin etc. (1997) Mol.Cell.Biol.17:4105-4113.

Bmi-1 albumen is the member of vertebrates polycomb complex, and has participated in Transcriptional Silencing.It comprises a plurality of binding sites of various polycomb composite assemblies.Therefore, Bmi-1 and its function fragment can be used as and prevent domain.Gunster etc. (1997) Mol.Cell.Biol.17:2326-2335; Hemenway etc. (1998) Oncogene16:2541-2547.

E2F6 albumen is the member of containing the polycomb complex of mammal Bmi-1, and is the transcription repressor that can collect RYBP, Bmi-1 and RING1A.The function fragment that comprises the E2F6 of amino acid/11 29-281 is used as the transcription repression domain.Therefore, E2F6 and its function fragment can be used as and prevent domain.Trimarchi etc. (2001) Proc.Natl.Acad.Sci.USA 98:1519-1524.

Eed1 albumen to small part is prevented and is transcribed by collection histone deacetylase (as HDAC2).Prevent activity to be present in this proteic N-and the C-stub area.Therefore, eed1 and its function fragment can be used as and prevent domain.(1999) Nature Genet.23:474-478 such as vander Vlag.

CTBP2 albumen is prevented by collection HPC2-polycomb complex to small part and is transcribed.Therefore, CTBP2 and its function fragment can be used as and prevent domain.Richard etc. (1999) Mol.Cell Biol.19:777-787.

Neuron-restrictive silencer factor is the protein of preventing neuronal specificity gene expression.Therefore, NRSF or its function fragment can be used as and prevent domain.Referring to for example U.S. Patent number 6,270,990.

Other prevents domain to comprise PLZF, BCL-6, BAZF, ZNF274, PRH, TEL, TGIF and G9A.

It will be understood by those skilled in the art that between DNA is in conjunction with territory and functional domain, to form in the fusion rotein (or encode its nucleic acid) repressor or be suitable as functional domain with the interactional molecule of repressor.Basically, can collect and prevent complex and/or target gene is had the domain of preventing that any molecule of preventing activity (for example histone deacetylation) all can be used as fusion rotein.

Other exemplary activation domain includes but not limited to: p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2.Referring to (2000) Mol.Endocrinol.14:329-347 such as for example Robyr; Collingwood etc. (1999) J.Mol.Endocrinol.23:255-275; Leo etc. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim.Pol.46:77-89; McKenna etc. (1999) J.Steroid Biochem.Mol.Biol.69:3-12; Malik etc. (2000) Trends Biochem.Sci.25:277-283; With (1999) Curr.Opin.Genet.Dev.9:499-504 such as Lemon.Other exemplary activation domain includes but not limited to: OsGAI, HALF-1, C1, AP1, ARF-5 ,-6 ,-7 and-8, CPRF1, CPRF4, MYC-RP/GP and TRAB1.Referring to (2000) Gene 245:21-29 such as for example Ogawa; Okanami etc. (1996) Gened Cells 1:87-99; Goff etc. (1991) GenesDev.5:298-309; Cho etc. (1999) Plant Mol.Biol.40:419-429; Ulmason etc. (1999) Proc.Natl.Acad.Sci.USA 96:5844-5849; Sprenger-Haussels etc. (2000) Plant J.22:1-8; Gong etc. (1999) Plant Mol.Biol.41:33-44; With (1999) Proc.Natl.Acad.Sci.USA 96:15 such as Hobo, 348-15,353.

Other transcription activating domain can be available from following albumen: ATF2, myc, GATA-1, GATA-3, NF-E2, Oct1, CTF1, Sp1, GR-AF1, zeste disappearance, HSF-1, p53, myoD and CAR β.

It will be understood by those skilled in the art that between DNA is in conjunction with territory and functional domain, to form in the fusion rotein (or encode its nucleic acid) activation domain or be suitable as functional domain with the interactional molecule of activation domain.In fact, can collect and activate complex and/or target gene is had the activation domain that any molecule that activates active (for example acetylation of histone) all can be used as fusion rotein.

For example, Gong You U.S. Patent application 2002/0115215 and 2003/0082552 and total WO02/44376 in insulator structure territory, location structure territory and chromatin remodeling albumen domain and/or the methyl binding domain protein as containing ISWI that is suitable as the fusion molecule functional domain described.

In another embodiment, DNA is blended in difunctional domain (BFD) in conjunction with territory (as Zinc finger domain).Difunctional domain is the transcriptional regulatory territory that activity depends on the BFD and second interaction of molecules.Second molecule can be the molecule that can influence any kind of BFD functional characteristic, includes but not limited to chemical compound, micromolecule, peptide, protein, polysaccharide or nucleic acid.Exemplary BFD is the ligand binding domain of estrogen receptor (ER).In the presence of estradiol, the ER ligand binding domain is as transcriptional activator; Yet, do not have estradiol but have tamoxifen or during the 4-trans-Hydroxytamoxifen, it is as transcription repressor.Another example of BFD is Thyroid Hormone Receptors (TR) ligand binding domain, is not having under the situation of part, and this ligand binding domain is as transcription repressor, and under the situation that has thyroxin (T3), it is as transcriptional activator.Another kind of BFD is glucocorticoid receptor (GR) (GR) ligand binding domain.In the presence of dexamethasone, this domain is as transcriptional activator; Yet in the presence of RU486, it is as transcription repressor.Another kind of exemplary BFD is the ligand binding domain of retinoic acid receptors.In the presence of its part all-trans retinoic acid, retinoic acid receptors has been collected many co-activation thing complex and activated transcription.Do not having under the situation of part, retinoic acid receptors can not be collected and transcribe the co-activation thing.Known other BFD of those skilled in the art.Referring to for example U.S. Patent number 5,834,266 and 5,994,313 and PCTWO 99/10508.

Another kind of functional domain derived from nuclear receptor is the domain that functional activity is regulated by the non-natural part.They usually are the mutant or the trim of natural generation receptor, are sometimes referred to as " convertible " domain.For example, some mutant of progesterone receptor (PR) can not interact with its native ligand, therefore can not be by the progesterone transcriptional activation.Yet some can be by activating in conjunction with micromolecule (example is the progesterone antagonist mifepristone) beyond the progesterone in these mutants.This non-natural but have the part of function to be called as hormone antagonist.Referring to for example United States Patent (USP) 5,364,791; 5,874,534; 5,935,934; Wang etc., (1994) Proc.Natl.Acad.Sci.USA 91:8180-8184; Wang etc. (1997) Gene Ther.4:432-441.

Therefore, the mifepristone dependency that comprises the endogenous gene that targeting DNA can be used for selecting in conjunction with the fusions of the PR ligand binding domain of the sudden change of territory (as ZFP), functional domain and this type activates or prevents, by design or select DNA in conjunction with the territory so that it is in conjunction with near selected gene in territory or the selected gene.This fusions also can comprise the degron domain.If this fusions contains activation domain, the mifepristone dependency that can obtain gene expression so activates; Prevent domain if this fusions contains, the mifepristone dependency that can obtain gene expression is so prevented.In addition, the polynucleotide of this fusion rotein of encoding are provided, the cell that the carrier that comprises this polynucleotide also is provided and has comprised this polynucleotide and carrier.The modification or the mutant form that it will be understood by those skilled in the art that receptor except that PR also can be used as the convertible structure territory.Referring to (1989) EMBO such as for example Tora J.8:1981-1986.

Host cell, carrier and promoter

In the enforcement of described method, in cell, express above-mentioned engineered zinc finger protein, this protein binding is transcribed with adjusting in target site.For example, albumen can be delivered to these proteic polynucleotide of maybe will encoding in the cell and be delivered in the cell, in cell, to express this albumen.This proteic dna molecular is delivered in the cell if will encode, and it can be transcribed into the mRNA molecule, and this mRNA molecule can produce this albumen through translation.Perhaps, the RNA molecule can be delivered in the cell, and translate this RNA molecule and produce this albumen.Known in the art with polynucleotide and the polypeptide delivery method in the cell, exemplary method has been listed in other place of the disclosure.Also can be by the following method in cell, express this albumen:, and select this nucleic acid to be stabilized to be integrated into chromosome or be stabilized and can be maintained at hereditarily cell line in the cell with coding this proteic nucleic acid transfection cell.In these cases, the protein expression of stable maintenance sequential coding can be induction type (as tetracycline-and the system of doxycycline-adjusting) or composing type.

Described compositions and method are used in any cell carries out transcriptional regulatory to any nucleotide sequence, and cell comprises the cell in cultured cell, primary cell, the organism and takes out, sends it cell of this organism back to after being delivered to albumen or these proteic polynucleotide of encoding the cell from organism.Aspect this, the transcribing of scalable endogenous and exogenous sequence.In order to regulate transcribing of endogenous sequence, one or more SEQ ID NO:1 copies can be inserted and wait to regulate in the endogenous sequence or near it.Be used for exogenous sequence targeting ground is inserted the total U.S. Patent Application Serial Number 10/912 of the exemplary method of cellular genome referring to U.S. Patent Application Publication No. 2003/0232410 (on December 18th, 2003), the open WO 03/87341 (on October 23rd, 2003) of PCT and submission on August 6th, 2004,932, its full content by with reference to including this paper in, is used for all purposes.

As mentioned above, disclosed method and compositions also can be used for regulating transcribing of exogenous sequence (as introducing the carrier that comprises the cDNA sequence of cell).For example cDNA can be cloned into and contain promoter, as the carrier of SR α promoter, so that transcribing of this cDNA sequence controlled by this promoter.In these cases, the carrier that will contain cDNA is introduced cell, and engineered ZFP as described herein (or polynucleotide of the engineered ZFP as described herein that encodes) is introduced same cell.Perhaps, the cDNA that will contain the polynucleotide copies of (or stable maintenance) coding of integration engineered ZFP as described herein introduces stable cell lines.Perhaps, the polynucleotide of the engineered ZFP or the engineered ZFP that encodes are introduced the cell that (or stable maintenance) of containing integration contains the polynucleotide sequence copy of cDNA.

In the cell of the stable integration sequence that contains the engineered ZFP of coding, can introduce many exogenous transcript units (target site that contains one or more ZFP separately) (as by transfection), to obtain the collaborative adjusting of exogenous transcript unit.Also one or more target sites can be introduced in these cells, be located at any amount of endogenous transcript unit upstream, inside or near, to obtain the collaborative adjusting of a plurality of genes.

Be used to the increase suitable carrier of certain extracellular endogenous nucleotide sequence and the promoter of transcribing that is used to regulate this exogenous nucleotide sequence known in the art.Exemplary promoter comprises SR α and CMV promoter.Available described albumen is directly regulated the transcribing of sequence that operability is connected in SR α promoter, because there is the copy of target site SEQ ID NO:1 in the SR α promoter.Can be by more SEQ ID NO:1 copies be introduced transcribing (as being used for proteinic overexpression) of SR α promoter realization higher level.And, can cause expression to be higher than the expression that obtains with the unmodified promoter by inserting one or more SEQ ID NO:1 copy modification SR α promoter (as the CMV promoter) in addition.

Described method and composition can be used for the cell of any kind, includes but not limited to prokaryotic cell, fungal cell, archeobacteria cell, plant cell, insect cell, zooblast, vertebrate cells, mammalian cell and people's cell.Those skilled in the art become known for the suitable cell line of protein expression, include but not limited to: COS, CHO is (as CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 is (as HEK293-F, HEK293-H, HEK293-T), perC6, noctuid (Spodoptera fugiperda) is coveted (Sf) and fungal cell such as yeast (Saccharomyces) in insect cell such as meadow, Pichia sp. (Pischia) and fission yeast (Schizosaccharomyces).Also can adopt offspring, variant and the derivant of these cell lines.

Exemplary promoter comprises SR α, CMV, phosphoglyceric kinase (PGK), people's ubiquitin C (UBC), EF-1 α (EF-1 α), herpes thymidine kinase (TK), SV40 is early stage and late promoter, human keratin-14 (K14) and rous sarcoma virus (RSV).

In some embodiments, available same engineered ZFP regulates two or more promoteres, as long as in the promoter or have one or more target sites of ZFP near it.This is particularly useful for the recombinant production of antibody, because the sequence of encoding heavy chain can be placed transcribing of first promoter to control down, what the sequence of coding light chain can be placed second promoter transcribes control down.These two kinds of promoteres can be identical, and perhaps they can be different promoteres.In other embodiments, the same promoter that contains two copies of varying number ZFP target site separately can be used for distinguishing and expresses the sequence that operability is connected in each promoter.Also can adopt two kinds of different promoters that contain the varying number target site separately.

It will be understood by those skilled in the art that and to design a ZFP, make it in conjunction with any predetermined target sequence; The ZFP that therefore may adopt this sequence of one or more copies and be incorporated into this sequence regulates and transcribes, to carry out (for example) overexpression.

Integrative nucleic acid and expression vector

In some embodiments, integrative nucleic acid coding fused polypeptide.In this case, nucleic acid clone can be gone in intermediary's carrier to be transformed into protokaryon or eukaryotic cell to duplicate and/or express.The intermediary's carrier that is used to store or operate integrative nucleic acid or produce fusion rotein can be (for example) prokaryotic vector (as plasmid), shuttle vector, insecticide carrier or viral vector.Also integrative nucleic acid can be cloned into expression vector, to give bacterial cell, fungal cell, protozoan cell, plant cell or zooblast, preferred mammal cell, more preferably people's cell.

The nucleic acid clone of encoding fusion protein can be gone into carrier, duplicate and/or express to be transformed into protokaryon or eukaryotic cell.Carrier can be prokaryotic vector such as plasmid, or shuttle vector, insecticide carrier or eukaryotic vector.Also the nucleic acid clone of coding ZFP can be gone in the expression vector, to give plant cell, zooblast, fungal cell, bacterial cell or protozoan cell, zooblast preferred mammal cell or people's cell.

For the gene or the nucleic acid of expression cloning, generally the sequence sub-clone of encoding fusion protein is gone into to contain in the expression vector that promoter transcribes with guidance.Well known suitable antibacterial and eukaryotic promoter, referring to for example Sambrook etc., " molecular cloning, laboratory manual " (Molecular Cloning, A Laboratory Manual) (the 2nd edition, 1989; The third edition, 2001); Kriegler, " gene transfer and expression: laboratory manual " (Gene Transfer andExpression:A Laboratory Manual) (1990); " newly organized molecular biology experiment guide " (Ausubel etc., the same).Be used to express ZFP bacterial expression system can available from (as) escherichia coli (E.coli), bacillus cereus (Bacillussp.) and salmonella (Salmonella) (Palva etc., Gene 22:229-235 (1983)).Can buy the test kit of these expression systems.Those skilled in the art know the eukaryotic expression system that is used for mammalian cell, yeast and insect cell, and they also can be buied.

Be used to instruct the promoter of the expression of nucleic acid of coded protein to depend on concrete application.For example, generally potent constitutive promoter is used for expressing and protein purification.On the contrary, when giving certain albumen in the body and be used for Gene regulation, adopt composing type or inducible promoter, this depends on this proteic concrete purposes.In addition, being used to give proteinic preferred promoter can be weak promoter, as HSV TK or have similar active promoter.Usually, this promoter also can comprise shifting the element that activation reacts, as anoxia response element, Gal4 response element, lac repressor response element and micromolecule control system such as tet-regulating system and RU-486 system (referring to for example Gossen and Bujard, PNAS 89:5547 (1992); Oligino etc., Gene Ther.5:491-496 (1998); Wang etc., Gene Ther.4:432-441 (1997); Neering etc., Blood 88:1147-1155 (1996); With Rendahl etc., Nat.Biotechnol.16:757-761 (1998)).

Except promoter, transcript unit that expression vector comprises or expression cassette generally contain all required other elements of express nucleic acid in protokaryon or eukaryotic host cell.Therefore, expression cassette generally contains operability and is connected in the promoter of nucleotide sequence of (for example) coding ZFP and the necessary signal of effective polyadenylation, tanscription termination, ribosome binding site or translation termination of (for example) transcript.The additional element of this box for example can comprise, enhancer and allos splicing signal.

According to proteinic required purposes, select to be used for the carrier that embodies in the hereditary information transporte to cells as in plant, animal, antibacterial, fungus, protozoacide etc., expressing (referring to following expression vector).The bacterial expression vector of standard comprises plasmid as the plasmid based on pBR322, pSKF, pET23D, and commercially available amalgamation and expression system such as GST and LacZ.Exemplary fusion rotein is maltose-binding protein " MBP." this fusion rotein helps protein purification.Also epi-position label such as c-myc, hemagglutinin (HA) or FLAG can be added recombiant protein, to be provided for separating, monitoring the method that makes things convenient for of expressing and monitor cell and Subcellular Localization.

The expression vector that contains the controlling element of eucaryon virus usually is used for carrier for expression of eukaryon, as SV40 carrier, papillomatosis poisonous carrier with derived from the carrier of Epstein-Barr virus.Other exemplary eukaryotic vector comprises pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE and can express proteic any other carrier under the guidance of following promoter: SV40 early promoter, SV40 late promoter, metallothionein promoter, molluscum contagiosum room oncovirus promoter, rous sarcoma virus promoter, polyhedrin promoter or the verified promoter that can be effective to eukaryotic cell expression.

Some expression systems contain the labelling that is useful on the selection stably transfected cell line, as thymidine kinase, hygromycin B phosphotransferase and dihydrofolate reductase.The high yield expressing system also is suitable, and as adopt baculovirus vector in insect cell, wherein protein coding sequence is under the guidance of polyhedrin promoter or other potent bacilliform virus promoter.

The gene and being used to of coding antibiotic resistance that the element that expression vector generally comprises is also included within the replicon that works in escherichia coli or other protokaryon antibacterial, be used for selecting carrying the antibacterial of recombiant plasmid inserts unique restriction site of the plasmid nonessential region of recombination sequence.

Produce to express a large amount of proteic antibacterials, mammal, yeast or insect cell line with the standard transfection method, use then these albumen of standard technique purification (referring to for example Colley etc., J.Biol.Chem.264:17619-17622 (1989); " protein purification guide " (Guide to Protein Purificaion), " Enzymology method " (Methods inEnzymology), the 182nd volume (Deutscher compiles, 1990)).The conversion of eucaryon and prokaryotic cell according to standard technique carry out (referring to for example Morrison, J.Bact.132:349-351 (1977); Clark-Curtiss and Curtiss, " Enzymology method " (Methods in Enzymology) 101:347-362 (volume such as Wu, 1983).

Can adopt any well-known process of the foreign nucleus nucleotide sequence being introduced host cell.They comprise adopts calcium phosphate transfection, polybrene (polybrene), protoplast fusion, electroporation, liposome, microinjection, naked DNA, plasmid vector, viral vector, episome and integrate body (integrative), and any other well-known process (referring to for example Sambrook etc., the same) of cloned genes group DNA, cDNA, synthetic DNA or other foreign heredity substance being introduced host cell.Used concrete genetic engineering method only needs at least a gene successfully to be introduced can express in the selected proteic host cell.

The nucleic acid and the cell of encoding fusion protein are sent

Can adopt based in virus and the conventional gene transfer method of non-virus the nucleic acid introducing cell (as mammalian cell) and target tissue encoding fusion protein and/or engineered ZFP.Also can adopt this method to give cell at external nucleic acid with encoding fusion protein.In some embodiments, the nucleic acid that gives encoding fusion protein carries out in the body or stripped (ex vivo) gene therapy application.The non-virus carrier delivery system comprise DNA plasmid, naked nucleic acid and with delivery vector such as liposome or the compound nucleic acid of poloxamer (poloxamer).The viral vector delivery system comprises DNA and RNA viruses, and they become episome or are integrated into genome after being delivered to cell.The summary of gene therapy method is referring to Anderson, Science 256:808-813 (1992); Nabel and Felgner, TIBTECH 11:211-217 (1993); Mitani and Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada etc., " microorganism and immunologic current theme " (Current Topics in Microbiology and Immunology) Doerfler and B  hm (volume) (1995); With Yu etc., Gene Therapy 1:13-26 (1994).

The non-viral delivering method of nucleic acid of engineered ZFP of encoding comprises electroporation, fat transfection, microinjection, biological projectile, virion, liposome, immunoliposome, polycation or lipid: the enhanced DNA picked-up of nucleic acid conjugates, naked DNA, artificial viral body and material.The fat transfection is referring to for example US 5,049,386, US 4,946,787 and US 4,897,355), lipofectin reagent is commercially available (as Transfectam ^TM, Lipofectin ^TM, Lipofectamine ^).The cation and the neutral lipid that are applicable to effective receptor identification fat transfection of polynucleotide comprise Felgner, and WO 91/17424, and WO 91/16024.Can be delivered to cell (exsomatize and give) or target tissue (giving in the body).

Lipid: nucleic acid complexes, the preparation that comprises target liposomes such as immune lipid complex be well known to those skilled in the art (referring to for example Crystal, Science 270:404-410 (1995); Blaese etc., Cancer Gene Ther.2:291-297 (1995); Behr etc., Bioconjugate Chem.5:382-389 (1994); Remy etc., BioconjugateChem.5:647-654 (1994); Gao etc., Gene Therapy 2:710-722 (1995); Ahmad etc., CancerRes.52:4817-4820 (1992); U.S. Patent number 4,186,183,4,217,344,4,235,871,4,261,975,4,485,054,4,501,728,4,774,085,4,837,028 and 4,946,787).

The system based on RNA or DNA viruses that is used to send the nucleic acid of encoding fusion protein and/or engineered ZFP has utilized with specific cells in the viral targeting body and with viral payload and has been transported to the method that the height of nuclear is evolved.Viral vector can directly give the patient (in the body), and perhaps available their extracorporeal treatment cells give the patient (exsomatizing) with the cell of modifying then.The conventional system based on virus of sending ZFP includes but not limited to: the retrovirus, slow virus, adenovirus, adeno associated virus, cowpox and the herpes simplex virus vector that are used for gene transfer.May be integrated into host genome with retrovirus, slow virus and adeno associated virus gene transfer method, usually cause the genetically modified long-term expression of inserting.In addition, in many different cell types and target tissue, observe high transduction efficiency.

Can be by mixing the potential target population change retrovirus tropism (tropism) of external envelope protein, expansion target cell.Slow virus carrier is the retroviral vector that can transduce or infect Unseparated Cell, generally produces high virus titer.Target tissue is depended in the selection of reverse transcription virus gene transfer system.Retroviral vector is grown terminal repetition the composition by cis acting, and its bale capacity is up to the external sequence of 6-10kb.Minimum cis acting LTR is enough to duplicate and package carrier, with LTR therapeutic gene is integrated into target cell then, so that permanent transgene expression to be provided.Extensively the retroviral vector that adopts comprise carrier based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), HIV (human immunodeficiency virus) (HIV) and combination thereof (referring to for example Buchscher etc., J.Virol.66:2731-2739 (1992); Johann etc., J.Virol.66:1635-1640 (1992); Sommerfelt etc., Virol.176:58-59 (1990); Wilson etc., J.Virol.63:2374-2378 (1989); Miller etc., J.Virol.65:2220-2224 (1991); PCT/US94/05700).

In the application of preferred transient expression fusion rotein, can adopt system based on adenovirus.Carrier based on adenovirus can be transduceed in many cell types very efficiently, and does not need cell division.Obtained with this carrier that height is tired and high level expression.Can in simple relatively system, produce this carrier in a large number.Adeno associated virus (" AAV ") carrier also is used for the target nucleic acid transducer cell, as at external generation nucleic acid and peptide, and be used in the body and stripped gene therapy method (referring to for example West etc., Virology 160:38-47 (1987); U.S. Patent number 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin.Invest.94:1351 (1994).Many documents comprise U.S. Patent number 5,173,414; Tratschin etc., Mol.Cell.Biol.5:3251-3260 (1985); Tratschin etc., Mol.Cell.Biol.4:2072-2081 (1984); Hermonat and Muzyczka, PNAS 81:6466-6470 (1984); With Samulski etc., the structure of reorganization AAV carrier has been described among the J.Virol.63:03822-3828 (1989).

In clinical trial, have at least six kinds of viral vector methods to can be used for gene transfer at present, the method that they utilize comprises with the gene that inserts auxiliary cell line replenishes the defective carrier, to produce the transduction material.

PLASN and MFG-S are example (Dunbar etc., the Blood85:3048-305 (1995) that is used for the retroviral vector of clinical trial; Kohn etc., Nat.Med.1:1017-102 (1995); Malech etc., PNAS 94:2212133-12138 (1997)).PA317/pLASN is the first kind of treatment carrier that is used for the gene therapy test.(Blaese etc., Science 270:475-480 (1995)).Observed transduction efficiency is 50% or higher in the MFG-S package carrier.(Ellem etc., Immunol Immunother.44 (1): 10-20 (1997); Dranoff etc., Hum.GeneTher.1:111-2 (1997).

Recombinant adeno-associated virus vector (rAAV) is an another kind of genes delivery system likely, and it is based on deficiency and non-pathogenic parvovirus 2 type adeno associated virus viruses.All carriers are all available from only keeping side joint in the terminal reverse multiple plasmid of the AAV of transgene expression cassette 145bp.Owing to be integrated in the genome of transducer cell, it is the key feature of this carrier system that efficient gene transfer and stable transgenic are sent.(Wagner etc., Lancet 351:91171702-3 (1998), Keams etc., Gene Ther.9:748-55 (1996)).

Can produce the replication defective recombinant adenoviral vector (Ad) that height is tired, they infect many different cell types easily.Engineered most of adenovirus vector substitutes Ad E1a, E1b and/or E3 gene with render transgenic; In people's 293 cells of the trans gene function that disappearance is provided, breed replication-defective vector subsequently.But the multiple types of organization of transduction comprises non-division, noble cells, as the cell in liver, kidney and the muscle in the Ad carrier body.Conventional Ad carrier has the capacity that carries greatly.An example using the Ad carrier in the clinical trial comprises the polynucleotide treatments (Sterman etc., Hum.Gene Ther.7:1083-9 (1998)) of carrying out antineoplastic immune with intramuscular injection.Other example that adenovirus vector is used for the gene transfer of clinical trial comprises Rosenecker etc., Infection 24:15-10 (1996); Sterman etc., Hum.Gene Ther.9:71083-1089 (1998); Welsh etc., Hum.Gene Ther.2:205-18 (1995); Alvarez etc., Hum.Gene Ther.5:597-613 (1997); Topf etc., Gene Ther.5:507-513 (1998); Sterman etc., Hum.Gene Ther.7:1083-1089 (1998).

With incasing cells form can host cells infected virion.This cell comprises 293 cells, ψ 2 cells or PA317 cell, 293 cells energy encapsidated adenovirus virus, and the PA317 cell can be packed retrovirus.Usually use the production cell line that nucleic acid carrier is packaged in the virion to produce the viral vector that is used for gene therapy.Carrier generally comprises packing and is integrated into the required minimum virus sequence of host (if feasible) subsequently, and other virus sequence expression cassette for the treatment of expressing protein that is encoded substitutes.The trans viral function of losing that provides of package cell line.For example, the AAV carrier that is used for gene therapy generally only has packing and is integrated into the genomic end of the necessary AAV of host genome and oppositely repeats (ITR) sequence.Viral DNA is packaged in the cell line that contains helper plasmid, this helper plasmid other AAV gene of encoding, i.e. rep and cap, but do not have the ITR sequence.Also infect this cell line as helper virus with adenovirus.Helper virus promote the AAV carrier duplicate with helper plasmid in the AAV expression of gene.Owing to lack the ITR sequence, can not pack a large amount of helper plasmids.Can reduce adenovirus by (for example) heat treatment and pollute, adenovirus is higher than AAV to heat treated sensitivity.Perhaps, can on plasmid, provide the adenovirus miscellaneous function.

In many gene therapies are used, need gene therapy vector be delivered to particular tissue type with high degree of specificity.Therefore, but the modification virus carrier makes it have specificity to given cell, virus capsid protein formation fusion rotein on this part and the viral outer surface by expressing part.Select part, make it have affinity the known receptor that is present on the cells of interest type.Han etc. for example, Proc.Natl.Acad.Sci.USA 92:9747-9751 (1995) report, can modify Moloney murine leukemia virus and transfer albumen with the people that expression is blended in gp70, this recombinant virus can infect some human breast cancer cell of expressing human EGF-R ELISA.It is right that this principle may extend to other virus-target cell, and wherein target cell is expressed a kind of receptor, and expressing viral comprises the fusion rotein of the part of this cell surface receptor.For example, can engineered filobactivirus, to present the antibody fragment (as Fab or Fv) that any selected cell receptor is basically all had the specificity binding affinity.Though foregoing description is applied to viral vector basically, same principle also can be applicable to non-virus carrier.Can engineered these carriers, make it contain the specificity picked-up sequence that helps the particular target cellular uptake.

Can be by giving single patient, generally be by delivery of gene treatment carrier in whole body administration (as intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or the topical application body, as described below.Perhaps, can with the carrier ex vivo delivered to cell, for example, in the single patient body, take out cell (drawing thing, biopsy samples) or universal donor hematopoietic stem cell as lymphocyte, bone marrow, then, after the cell of carrier is mixed in selection, cell is replanted into the patient usually.

Those skilled in the art know be used for diagnosing, the isolated cells transfection (as by transfectional cell is infused into host organisms again) of research or gene therapy.In some embodiments, cell separation is from the object organisms body, and with nucleic acid (gene or cDNA) transfection, infusion returns in the object organisms body (as the patient) again.Those skilled in the art know the various cell types that are applicable to the transfection of exsomatizing (referring to for example Freshney etc., " animal cell culture, basic fundamental handbook (Cultureof Animal Cells, A Manual of Basic Technique) (the 3rd edition, 1994) in the list of references of), wherein quoting about how separating and cultivate the argumentation of patient's cell).

In one embodiment, stem cell is used for the stripped method of cell transfecting and gene therapy.The advantage that adopts stem cell is that they can perhaps can be introduced in the mammal (as cell donor) in vitro differentiation for other cell type, can move in the bone marrow subsequently.The method that makes the CD34+ cells in vitro be divided into important clinically immunocyte type with cytokine such as GM-CSF, IFN-γ and TNF-α is known (referring to Inaba etc., J.Exp.Med.176:1693-1702 (1992)).

Separate with known method and to be used to the stem cell of transduceing and breaking up.For example, do not want cell with combination, antibody elutriation medullary cell as CD4+ and CD8+ (T cell), CD45+ (general B cell (panB cell)), GR-1 (granulocyte) and Iad (antigen presenting cell of differentiation), thereby the stem cell in the separation medullary cell (referring to Inaba etc., J.Exp.Med.176:1693-1702 (1992)).

Also can directly give organism and carry out cells in vivo transduction containing the carrier (as retrovirus, adenovirus, liposome etc.) for the treatment of nucleic acid.Perhaps, can give naked DNA.Carry out administration by the approach that is generally used for molecule is finally contacted with blood or histiocyte, these approach include but not limited to: injection, infusion, topical application and electroporation.Can obtain to be fit to give the method for this nucleic acid, they are well known to those skilled in the art, though can adopt more than one approach to give concrete compositions, certain concrete approach usually can provide than the more direct and more effective reaction of other approach.

Pharmaceutically acceptable carrier part is by the concrete compositions that gives, and the concrete grammar decision that is used to give said composition.Therefore, can adopt various suitable pharmaceutical compositions dosage forms, (referring to for example " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences), the 17th edition, 1989) as described below.

Can the DNA construction be introduced in the genome of required plant host by various routine techniquess.The summary of these technology referring to for example Weissbach and Weissbach " molecular biology of plants method " (Methods for PlantMolecular Biology) (1988, Academic Press, N.Y.) VIII chapter, 421-463 page or leaf; With Grierson and Corey, " molecular biology of plants " (Plant Molecular Biology) (1988, the 2 editions), Blackie, London, 7-9 chapter.For example, can adopt such as in the genomic DNAs of technology with the direct introduced plant cell of DNA construction such as the electroporation of plant protoplast and microinjection, perhaps available biological projectile method is bombarded the direct introduced plant tissue of DNA construction (referring to (1987) Nature 327:70-73 such as for example Klein) as dna particle.Perhaps, the DNA construction can make up with suitable T-DNA side joint district and introduce in conventional Agrobacterium tumefaciens (Agrobacterium tumefaciens) host's carrier.Describe the transformation technology of Agrobacterium tumefaciens-mediation in the scientific literature in detail, comprise arms (disarming) and adopt two carriers.Referring to (1983) Proc.Nat ' l.Acad.Sci.USA 80:4803 such as (1984) Science233:496-498 such as for example Horsch and Fraley.When adopting double T dna vector (Bevan (1984) Nuc.Acid Res.12:8711-8721) or being total to cultivating method (Horsch etc. (1985) Science227:1229-1231) with the bacterial infection cell, Agrobacterium tumefaciens host's virulence function will instruct to be inserted construction and adjacent marker thing in the plant cell dna.Usually, with the engineered dicotyledon of edaphic bacillus (Agrobacterium) conversion system (Bevan etc. (1982) Ann.Rev.Genet 16:357-384; Rogers etc. (1986) MethodsEnzymol.118:627-641).The edaphic bacillus conversion system also can be used for DNA is transformed and transfers in monocotyledon and the plant cell.Referring to (1984) EMBO J 3:3039-3041 such as Hernalsteen; Hooykass-VanSlogteren etc. (1984) Nature 311:763-764; Grimsley etc. (1987) Nature 325:1677-179; Boulton etc. (1989) Plant Mol.Biol.12:31-40; With (1991) Plant Physiol.95:426-434 such as Gould.

Other gene transfer and conversion method include but not limited to: by calcium-, Polyethylene Glycol (PEG)-or the picked-up of the naked DNA of electroporation-mediation carry out protoplast transformation (referring to (1984) EMBO J 3:2717-2722 such as Paszkowski, Potrykus etc. (1985) Molec.Gen.Genet.199:169-177; Fromm etc. (1985) Proc.Nat.Acad.Sci.USA 82:5824-5828; And Shimamoto (1989) Nature 338:274-276) and the electroporation of plant tissue (D ' Halluin etc. (1992) Plant Cell 4:1495-1505).Be used for other method that plant cell transforms and comprise that the DNA picked-up (Kaeppler etc. (1990) Plant Cell Reporter 9:415-418) of microinjection, carborundum mediation and microparticle bombardment are (referring to (1988) Proc.Nat.Acad.Sci.USA 85:4305-4309 such as Klein; With (1990) Plant Cell 2:603-618 such as Gordon-Kamm).

Can cultivate the plant transformed cell that produces by any above-mentioned transformation technology, have the whole strain plant of transformed gene type and desired phenotype with regeneration.This regeneration techniques depends on handles the certain plants hormone in the tissue growth culture medium, generally depend on antimicrobial and/or the herbicide labelling introduced with required nucleotide sequence.Evans, etc., " protoplast separates and cultivates " (Protoplasts Isolation and Culture), " culture plant cell handbook (Handbook of Plant Cell Culture), 124-176 page or leaf, Macmillian Publishing Company, New York, 1983; And Binding, " plant, plant protoplast regeneration " (Regeneration of Plants, PlantProtoplasts), and the 21-73 page or leaf, CRC Press, the Berkeley village, 1985 have described from the protoplast regeneration plant of cultivating.Regeneration also can be available from plant callus, explant, organ, pollen, embryo or its part.Usually, among (1987) Ann.Rev.of Plant Phys.38:467-486 such as Klee this regeneration techniques has been described.

The nucleic acid of introduced plant cell is used in and produces required feature on any basically plant.Available nucleic acid construct thing of the present invention and engineered each kind of plant of above-mentioned various method for transformation and plant cell system make it have required physiology as herein described and agricultural economy feature.In some embodiments, being used for engineered target plant and plant cell includes but not limited to: unifacial leaf and dicotyledon, as crops, comprise cereal crops (as Semen Tritici aestivi, corn, rice, Semen setariae, Fructus Hordei Vulgaris), fruit crop (as Fructus Lycopersici esculenti, Fructus Mali pumilae, pears, Fructus Fragariae Ananssae, orange), forage crop (as Herba Medicaginis), root vegetables crop (as Radix Dauci Sativae, Rhizoma Solani tuber osi, Radix Betae, Rhizoma Dioscoreae), leaf vegetables crop (as Caulis et Folium Lactucae sativae, Herba Spinaciae); Flowering plant (as petunia, Flos Rosae Rugosae, Flos Chrysanthemi), coniferous tree and pinaster (as pinaster, fir, PiceameyeriRehd. Et Wils.); The used plant of plant scrubbing (as heavy metal accumulation plant); Plant (as arabidopsis (Arabidopsis)) is used in oil crop (as Helianthi, Semen Brassicae campestris) and experiment.Therefore, described method and composition can be widely used in each kind of plant, includes but not limited to Asparagus (Asparagus), Avena (Avena), Btassica (Brassica), Citrus (Citrus), Citrullus (Citrullus), Capsicum (Capsicum), Cucurbita (Cucurbita), Hu Luobu belongs to (Daucus), Glycine (Glycine), Hordeum (Hordeum), Lactuca (Lactuca), Fructus Lycopersici esculenti belongs to (Lycopersicon), Malus (Malus), cassava (Manihot), Nicotiana (Nicotiana), Oryza (Oryza), Persea (Persea), Pisum (Pisum), pear (Pyrus), Prunus (Prunus), Rhaphanus (Raphanus), Secale (Secale), Solanum (Solanum), sorghum (Sorghum), Triticum (Triticum), Vitis (Vitis), the kind of Vigna (Vigna) and Zea (Zea).

One skilled in the art will know that, expression cassette is stable mix transgenic plant and confirm to operate after, can be introduced into other plant by sexual hybridization (sexual crossing).Can adopt any in many standard cultivation technology, this depends on the kind of preparing hybridization.

Can by the characterized selecting or screen the marker gene coding that exists on the transforming DNA in the engineered phyteral with separate plant transformed cell, callus, tissue or plant.For example, can select by the phyteral of culturing engineering transformation on the culture medium of antibiotic that contains amount of suppression or herbicide, the gene constructs of conversion produces resistance to above-mentioned condition of culture.And, also can be by screening the activity identification plant transformed and the plant cell of any witness marking thing gene (as β-glucuronidase, luciferase, B or C1 gene) that may exist on the recombinant nucleic acid construction.Those skilled in the art know this selection and screening technique.

Also can adopt physiology and biochemical method to identify and contain plant or the plant cell conversion product that inserts gene constructs.These methods include but not limited to: the Southern analysis or the pcr amplification that 1) are used to detect or measure recombinant DNA insert structure; 2) be used to detect and check Northern trace, S1 RNA enzyme protection, primer extension or the reverse transcriptase-pcr amplification of the rna transcription thing of gene constructs; 3) enzyme test of detection enzyme or ribozyme activity, wherein said gene outcome have the gene constructs coding; 4) gel electrophoresis of protein, Western engram technology, immunoprecipitation or enzyme linked immune assay, wherein the gene constructs product is a protein.Also other technology be can adopt, the existence or the expression of recombination to construct thing in specified plant organ and the tissue detected as in situ hybridization, enzyme dyeing and immunostaining.Those skilled in the art know the method for carrying out all these tests.

Can observe the effect that adopts methods described herein to carry out genetic manipulation by the Northern trace that (for example) separates the RNA (as mRNA) that knits from interest groups.Usually, if mRNA content increases, the expression speed ratio that can infer corresponding endogenous gene was high in the past.Can adopt other method of measuring gene activity.Can adopt dissimilar enzyme tests, this depends on the increase of used substrate and detection reaction product or by-product or the method for minimizing.In addition, can pass through immuno-chemical method, i.e. ELISA, RIA, EIA and other test based on antibody well known to those skilled in the art is as the level of electrophoresis detection test (with dyeing or Western trace) mensuration expressing protein.Transgenic can be in some plant tissues or certain stage of development selective expression, and perhaps, transgenic can be in all plant tissues basically, express in the whole life basically.Yet, also can adopt any combination expression way.

The present invention also comprises the seed of above-mentioned transgenic plant, and wherein said seed contains described transgenic or gene constructs.The present invention also comprises offspring, clone, cell line or the cell of above-mentioned transgenic plant, and wherein said offspring, clone, cell line or cell have described transgenic or gene constructs.

Delivery vector

Giving polypeptide compound, is to guarantee that this polypeptide can pass cytoplasma membrane as the key factor of fusion rotein, or intracellular region chamber such as nuclear film.Cell membrane is formed by lipoprotein is double-deck, and it allows micromolecule nonionic lipophilic compound freedom penetrating, does not allow polar compound, macromole and treatment or diagnostic substances penetrating in essence.Yet, some albumen and other chemical compound such as liposome have been described, they can cross-cell membrane transhipment polypeptide.

For example " film transhipment polypeptide " has both sexes or the hydrophobic amino acid subsequence that can transport carrier as film.In one embodiment, homeodomain protein can the cross-cell membrane transhipment.But the shortest internalization peptide Antennapedia that finds homeodomain protein is the 3rd spiral on this albumen, from amino acid position 43 to 58 (referring to for example Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)).Another subsequence h (hydrophobic) domain of finding signal peptide has similar cell membrane transporter feature (referring to for example Lin etc., J.Biol.Chem.270:14255-14258 (1995)).

The example that can be connected in protein, helps the peptide sequence of protein uptake in the cell includes but not limited to: proteic 11 amino acid peptides of the tat of HIV; Peptide sequence (referring to Fahraeus etc., Current Biology 6:84 (1996)) corresponding to 20 residues of the proteic aminoacid 84-103 of p16; The 3rd spiral of the homeodomain of 60 amino acid longs of Antennapedia (Derossi etc., J.Biol.Chem.269:10444 (1994)); The h district of the h district of signal peptide such as Kaposi fibroblast growth factor (K-FGF) (Lin etc., the same); Or the VP22 translocation domain of HSV (Elliot and O ' Hare, Cell 88:223-233 (1997)).Also other appropriate chemical part chemistry that improves cellular uptake can be connected in ZFP.Film translocation domain (being the internalization domain) also can be selected from the library of randomization peptide sequence.Referring to (2003) Molecular Therapy 7 (5): S461 such as for example Yeh, summary #1191.

Lps molecule also can cross-cell membrane transhipment polypeptide.This molecule (being called " two toxin ") is usually by two parts at least: transhipment/form in conjunction with territory or polypeptide and isolating toxin structure territory or polypeptide.Usually, translocation domain or polypeptide are incorporated into cell receptor, then toxin are transported in the cell.Use several bacteriotoxins, comprise bacillus perfringens (Clostridium perfringens) ι toxin, diphtheria toxin, diphtherotoxin (DT), pseudomonas (Pseudomonas) exotoxin A (PE), pertussis toxin, PT (PT), anthrax bacillus (Bacillus anthracis) toxin and pertussis adenyl cyclase (CYA) with delivery of peptides in cell cytosol, become inside or amino terminal fusions (Arora etc., J.Biol.Chem., 268:3334-3341 (1993); Perelle etc., Infect.Immun., 61:5147-5156 (1993); Stenmark etc., J.Cell Biol.113:1025-1032 (1991); Donnelly etc., PNAS 90:3530-3534 (1993); Carbonetti etc., Abstr.Annu.Meet.Am.Soc.Microbiol.95:295 (1995); Sebo etc., Infect.Immun.63:3851-3857 (1995); Klimpel etc., PNAS U.S.A.89:10277-10281 (1992); With Novak etc., J.Biol.Chem.267:17186-17193 1992)).

Can adopt the fusion rotein of this peptide sequence cross-cell membrane transhipment ZFP and other type.Peptide sequence can be blended in transit sequence easily or use its derivatization.Usually, the part as fusion rotein provides transit sequence.Randomly, available joint is connected in transit sequence the remainder of fusion rotein.Can adopt any suitable joint, as peptide linker.As mentioned above.

Also can fusion rotein be introduced zooblast, the preferred mammal cell by liposome and liposome derivant such as immunoliposome.Term " liposome " refers to the vesicle be made up of one or more layers double-layer of lipoid of entad arranging, and it is wrapped in water.Water generally contains to be prepared to be delivered to the chemical compound of cell, as comprises degron domain and the ZFPDNA fusion rotein in conjunction with the territory.

Liposome and plasma membrane merge, thereby protein is discharged in the endochylema.Perhaps, liposome engulfed or the transhipment vesicle in by cellular uptake.In case in endosome or phagosome, liposome is degraded or is merged with this film of transporting vesicle, and discharges its content.

In carry out the existing method that medicine sends by liposome, liposome finally becomes can be penetrating and discharge the chemical compound (being fusion rotein in this case) of parcel at target tissue or cell.Send in order to carry out whole body or tissue specificity, can finish by (for example) passive mode and send, wherein liposome is degraded in time by the effect of various substance in vivos.Perhaps, active medicine discharges the permeability changes that comprises with drug-induced liposome vesicle.Can make up liposome membrane, make them become unstable when environment becomes acidity near liposome membrane (referring to for example PNAS 84:7851 (1987); Biochemistry 28:908 (1989)).When liposome during by the target cell endocytosis, for example, they become unstable and discharge its content.This stable process that goes is called fusant formation.DOPE (DOPE) is the basis of many " fusant formation " system.

These liposomees generally comprise protein and lipid composition, as neutrality and/or cation lipid, randomly comprise receptor identification molecule, as in conjunction with the predetermined cell surface receptor in territory or the antibody of part (as antigen).Available prepared in various methods liposome is as Szoka etc., Ann.Rev.Biophys.Bioeng.9:467 (1980), U.S. Patent number 4,186,183,4,217,344,4,235,871,4,261,975,4,485,054,4,501,728,4,774,085,4,837,028,4,235,871,4,261,975,4,485,054,4,501,728,4,774,085,4,837,028,4,946,787, PCT publication number WO 91 17424, Deamer and Bangham, Biochim.Biophys.Acta 443:629-634 (1976); Fraley etc., PNAS 76:3348-3352 (1979); Hope etc., Biochim.Biophys.Acta 812:55-65 (1985); Mayer etc., Biochim.Biophys.Acta 858:161-168 (1986); Williams etc., PNAS85:242-246 (1988); " liposome " be (Ostro (volume), 1983, the 1 chapters) (Liposome); Hope etc., Chem.Phys.Lip.40:89 (1986); Gregoriadis, " liposome technology " (Liposome Technology) (1984) and Lasic, " liposome: acquire application " (Liposome:from Physics to Applications) (1993) from physics) described.Suitable method for example comprises: supersound process, extruding, high pressure/homogenate, microfluidization, detergent dialysis, the inductive small liposome vesicle of calcium merge and the ether fusion method known all these methods of those skilled in the art.

In some embodiments, need be with the targeting that special targeting moieties such as concrete cell type, tissue is carried out liposome.The targeting that carries out liposome with various targeting moieties (as part, receptor and monoclonal antibody) has been described.Referring to for example U.S. Patent number 4,957,773 and 4,603,044.

The example of targeting moiety comprises the tumor associated antigen monoclonal antibody specific, as prostatic cancer specific antigen and MAGE.Also can come target tumor as the activation of ras or c-erbB2 or the gene outcome of overexpression generation by detecting oncogene.In addition, many tumors are expressed the antigen of expressing usually in fetal tissue, as alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA).Available various virus antigen such as hepatitis B virus core antigen and surface antigen (HBVc, HBVs), hepatitis C antigen, Epstein-Barr virus antigen, 1 type human immunodeficiency virus (HIV1) and human papillomavirus antigen targeting viral infection position.The surface molecular such as the integrin (as VCAM-1) of expressing with inflammation part, the Molecular Detection inflammation of selecting protein receptor specific recognition such as (as ELAM-1).

Can adopt the standard method that targeting substance is coupled to liposome.These methods generally include lipid composition, mix liposome as PHOSPHATIDYL ETHANOLAMINE, and this lipid composition can be by connecting targeting substance or deutero-lipophilic compound, as the deutero-bleomycin activation of lipid.The liposome that available (for example) mixes protein A make up antibody target liposome (referring to Renneisen etc., J.Biol.Chem., 265:16337-16342 (1990) and Leonetti etc., PNAS87:2448-2451 (1990).

Dosage

In the treatment of content of the present invention was used, the dosage that gives the patient should be enough to produce in time useful therapeutic response in the patient.In addition, concrete dosage can be used for the phenotypic alternation of determination experiment in being provided with, as in functional genomics research and cell or animal model.Can be by effectiveness and the K of used concrete ZFP _d, target cell the long-pending and patient's situation of nucleome, and patient's to be treated body weight or body surface area are determined dosage.The dosage size also depends on existence, characteristic and the degree of the side effect of following when particular compound or carrier give concrete patient.

For example, the maximum therapy effective dose of about 99% ZFP that calculates when being incorporated into target site is less than every cell about 1.5 * 10 ⁵～1.5 * 10 ⁶Individual specific Z FP molecule copy.This bound water ZFP number of each cell at ordinary times is calculated as follows, and adopts the nuclear volume of HeLa (about 1000 μ m ³Or 10 ^-12L; Cell Biology, (Altman and Katz compile (1976)).Because the HeLa nucleus is relatively large, need recomputate this dosage number with the volume of target cell nuclear.This calculating does not consider that other site is to the bonded competition effect of ZFP yet.This calculates also supposition, and all ZFP are positioned nuclear basically.With 100 * K _dValue calculate about 99% and be incorporated into target site, with 10 * K _dValue calculate about 90% and be incorporated into target site.In the present embodiment, K _d=25nM

ZFP+ target site  complex

That is DNA+ protein  DNA: protein complex

K _d=[DNA] [protein]

[DNA: protein complex]

When 50%ZFP in conjunction with the time, K _d=[protein]

Therefore working as [protein]=25nM and nucleome long-pending is 10 ^-12During L

[protein]=(25 * 10 ^-9Mole/L) (10 ^-12L/ nuclear) (6 * 10 ²³Molecule/mole)

=15,000 molecule/nuclear (50% in conjunction with time)

When 99% target spot is combined; 100 * K _d=[protein]

100 * K _d=[protein]=2.5 μ M

(2.5 * 10 ^-6Mole/L) (10 ^-12L/ nuclear) (6 * 10 ²³Molecule/mole)

=about 1,500,000 molecule/nuclear (target site 99% in conjunction with time).

Also can be by counting the suitable dose of the Mean Speed calculation code ZFP Expression of Fusion Protein carrier of ZFP degraded from the Mean Speed of promoter expression ZFP and cell.In some embodiments, can adopt the HSV TK promoter of weak promoter such as wild type or sudden change, as mentioned above.Calculate Gamma Magnitude fusion rotein dosage by counting used concrete proteic molecular weight.

In order to determine to give proteinic effective dose in disease treatment or the prevention, the doctor has estimated the blood circulation blood plasma level of the protein or this proteic nucleic acid of encoding, by the genotoxic potential that this albumen causes, and the output of progression of disease and this proteic antibody.Can finish administration by single dose or divided dose.

Pharmaceutical composition and administration

Can directly give the patient to carry out target gene adjusting, targeting DNA cutting and/or reorganization and treatment or prophylactic applications, for example cancer, ischemia, diabetic retinopathy, degeneration of macula, rheumatoid arthritis, psoriasis, HIV infection, sicklemia, Alzheimer's disease, muscular dystrophy, neurodegenerative disease, angiopathy, cystic fibrosis, apoplexy etc. with fusion rotein and these proteic expression vectors of coding.The example of the quenchable microorganism of ZFP gene therapy comprises pathogen, as chlamydia, rickettsia, mycobacteria, staphylococcus, streptococcus, Diplococcus pneumoniae, meningococcus and conococci, klebsiella, Bacillus proteus, husky thunder bacterium, pseudomonas, legionella (legionella), diphtheria corynebacterium, salmonella, bacillus cereus, cholera bacteria, clostridium tetani, bacillus botulinus, anthrax bacillus, plague bacillus, leptospira and Lyme disease antibacterial; Infectious fungus is as aspergillosis (Aspergillus), candida mycoderma (Candida); Protozoacide such as sporozoon (as plasmodium (Plasmodia)), rhizopod (as entamoeba (Entamoeba)) and flagellate (trypanosoma (Trypanosoma), Leishmania (Leishmania), trichomonacide (Trichomonas), giardia lamblia stiles (Giardia) etc.); Viral disease such as hepatitis (hepatitis A, hepatitis B or hepatitis C), herpesvirus is (as VZV, HSV-1, HSV-6, HSV-II, CMV and EBV), HIV, Ai Bola virus, adenovirus, influenza virus, banzi virus, ECHO virus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, Measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, human papillomavirus, poliovirus, rabies virus and arboviral encephalitides virus etc.

Treat effective dose by any approach that is generally used for protein or nucleic acid are finally contacted with tissue to be treated.With any suitable form, preferably give fusion rotein or code nucleic acid with pharmaceutically acceptable carrier.Can obtain to give the appropriate method of these instrumentalities, they are well known to those skilled in the art, though can adopt more than one approach to give concrete compositions, certain concrete approach usually can provide than the more direct and more effective reaction of other approach.

Pharmaceutically acceptable carrier part is by the concrete compositions that gives, and the concrete grammar decision that is used to give said composition.Therefore, can adopt various suitable pharmaceutical compositions dosage forms (referring to for example " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences), the 17th edition, 1985)).

Can be with making aerosol (can with its " atomizing ") separately or with the fusion rotein of other suitable ingredients combination or its code nucleic acid, to pass through inhalation.Aerosol can be packed into the pressurization the propellant accepted in, as dichlorodifluoromethane, propane, nitrogen etc.

Be applicable to the gastrointestinal tract external administration, dosage form as intravenous, intramuscular, Intradermal and subcutaneous route comprises aqueous and non-aqueous isotonic sterile injection solution and aqueous and non-aqueous sterile suspension, can contain antioxidant, buffer, antibacterial in the described injection solution and make the isoosmotic solute of blood of this dosage form and receptor to be treated, described sterile suspension can comprise suspending agent, solubilizing agent, thickening agent, stabilizing agent and antiseptic.Can by give in (for example) intravenous infusion, oral, local, intraperitoneal, intravesical or the sheath described compositions or.Can be at unit dose or multiple dose sealed container, as providing compound dosage forms in ampoule and the bottle.Can be from sterilized powder, granule and the preparation tablets injection solution and the suspension of aforesaid kind.

Use

Described albumen also can externally be used.For example engineered ZFP (or transcribing/translate lysate) can with (as) polynucleotide (as plasmid) in link coupled transcribing/translation system hatch, with in the external protein production of carrying out.

In other embodiments, the target site of engineered ZFP can be placed on the both sides of endogenous or exogenous coded sequence.In this case, engineered ZFP (optional be blended in (as) insulator structure territory; Referring to for example WO 01/02553; WO 02/44376) with combining of target site can protect coded sequence be not subjected to (as) influence of position effect and heterochromatinization.

Described method can with existing method (as in the amplification of dihydrofolate reductase gene flanking sequence based on the selection of methotrexate) the collaborative use, with the protein expression that obtains based on transcribing of template number that improves and higher level to improve the standard.

In other embodiments, can introduce the target sequence of engineered ZFP, make itself and another transcriptional regulatory molecule adjacent or overlapping, so that further regulate and control its adjusting with engineered ZFP with its binding site.Can adopt the engineered ZFP that is blended in transcription activating domain or transcription repression domain, also can adopt the ZFP that is not blended in functional domain, but the combination that other regulates molecule is regulated in its space.

The endogenous gene inactivation is to help protein production

In some embodiments, make the functionally inactive of one or more cytogene products, to improve proteinic level and/or the quality that produces with methods described herein and compositions.Can by (for example) destroy the encoding gene product endogenous cellular gene one or more allele (as " knocking out ") by gene and/or as have a U.S. Patent number 6,534,261 describedly make the gene function inactivation by being enough to transcribing of described endogenous cellular gene.

Can insert in the cellular genome by mutation (as chemistry or radiation-induced) or with exogenous DNA sequence and destroy endogenous cellular gene, randomly select required mutant cell subsequently.The insertion of exogenous sequence can be (behind retroviral infection or cells contacting dna molecular) or (as U.S. Patent number 5,614,396) of targeting at random.Also can cut genomic DNA and realize that with the combination of introducing exogenous DNA the targeting of exogenous sequence inserts by targeting.By with reference to will in fit into the total PCT WO2005/014791 of this paper and U.S. Provisional Patent Application 60/702,394 (submission on July 26th, 2005) and be respectively targeting and insert the method and composition that exogenous sequence provides homology dependency and homology dependent/non-dependent method.In addition, PCT WO 2005/014791 also discloses by the terminal method of carrying out targeted mutagenesis that is connected of targeting DNA cutting and non-homology subsequently.

For example, can make one or more genes (or its function) inactivation of apoptosis involvement process.The exemplary gene of apoptosis involvement sees Table 1.

Table 1

Guang winter enzyme 1

Guang winter enzyme 2

Guang winter enzyme 3

Guang winter enzyme 4

Guang winter enzyme 5

Guang winter enzyme 6

Guang winter enzyme 7

Guang winter enzyme 8

Guang winter enzyme 9

Guang winter enzyme 10

Guang winter enzyme 11

Guang winter enzyme 12

Guang winter enzyme 13

Guang winter enzyme 14

bax

bak

bik

APAF-1 (apoptosis body associated factor 1)

c-jun

Lactic acid dehydrogenase

Cytochrome c

Can make the negative gene inactivation of regulating cell cycle progression of product.The exemplary gene that participates in Cycle Regulation sees Table 2.

Table 2

p16

p19

Rb (retinoblastoma albumen)

p53

p73

Telomerase

Because some proteic biological activity can be influenced by its glycosylation mode, so may need to make its product to participate in some gene inactivation of protein glycosylation in some cases.Participate in glycosylated exemplary gene and see Table 3.

Table 3

A-1, the 6-fucosyltransferase

CMP-N-n acetylneuraminic acid n hydroxylase

The cmp sialic acid hydroxylase

Glucosamine-6-phosphate isomerase

Can make coding infect the gene inactivation of receptor (as virus or bacterial receptor) to increase protein output.For example, can make the gene inactivation of encoding murine piconavirus (MVM) receptor.

In some cases, may need to integrate the exogenous dna molecular of coding transgenic and the selectable marker genetically modified cell of integration (as be used to select to contain).If the selected marker thing is a genome encoding that need to integrate the cell of exogenous dna molecular, may need to make the endogenous gene inactivation of this selected marker thing of coding so.Inactivation can be part inactivation (so that reducing the level that produces label) or complete deactivation (so that not producing label).So the exemplary marker gene of inactivation sees Table 4.

Table 4

Dihydrofolate reductase

Glutamine synthetase

Hypoxanthine phosphoribosyltransferase

The gene inactivation that can make the encoding proteins enzyme is to increase protein output.Exemplary protease sees Table 5.

Table 5

Serine protease

Elastoser

Collagenase

Activator of plasminogen

For safety and obedience adjusting requirement, can make the gene inactivation of coding Protein virus (PRP) and/or false Protein virus (pseudo-prison).False Protein virus comprises that for example PRNP/PRP, Protein virus sample protein D oppel (PRND) and Protein virus shade (Shadow of Prion) are (SPRN).Referring to (1986) Science 233:364-367 (PRNP) such as for example Liao; (2003) Gene 314:89-102 (SPRN) such as Lu etc. (2000) Biochemistry 39:13575-13583 (PRND) and Premzl.

At the cell that is used for producing antibody molecule, may need to make the proteic gene inactivation of the coding antibodies that produces.For example, available any method as herein described makes the antigenic gene inactivation of encoding antibody identification.

Other cell processes that can influence protein output and quality for example comprises: duplicate, transcribe, RNA processing, translation, amino acid bio are synthetic, cellular metabolism, protein folding, protein degradation, protein transport, stress and plasmid copy number control.The just adjusting or the negative adjusting (comprising that inactivation or gene " knock out ") that should be understood that the gene that participates in said process can be used for improving proteinic output and/or the quality of expressing with methods described herein and compositions.

Embodiment

It is in order to illustrate but not the theme of requirement for restriction right that following examples are provided.

Embodiment 1: make up SR α promoter

Produce the SV40 early promoter by PCR from plasmid pRL-SV40 (Promega), it comprises the base pair 80-449 of pRL-SV40 sequence, and mixes the HindIII site at 3 ' end of promoter.Base 80-88 is 2392/00 and the 9bp target site of 2932/10ZFP.The Bpu10I restriction site is added in the PCR primer, make its upstream that is positioned at the ZFP binding site just, to help being cloned into subsequently the main chain carrier pcDNA3.1/zeo that has removed original CMV promoter.

Produce the R-U5 composite sequence by PCR from plasmid pDrive01-NSE (r)-RU5 v02 (Invivogen), it comprises the base pair 1774-2066 of plasmid sequence, comprises the HindIII restriction site in 5 ' primer binding site.The EcoRI restriction site is added 3 ' PCR primer, to help being cloned into the main chain carrier.

Connect SV40 and RU5 sequence with its HindIII site separately, form the SV40-RU5 fusions.The sequence of this hybrid promoters (SEQ ID NO:9) is seen Fig. 1, and the line part is the ZFP target site.

Embodiment 2: engineered zinc finger protein is to be incorporated into the target site in the SR α promoter

In order to increase the protein expression of coding region under the control of SR α promoter, synthetic three refer to zinc finger protein SBS2392/00 (according to total United States Patent (USP) 6,453,242 and 6,534,261 described methods), with the target sequence in conjunction with 9 nucleotide in the SR α promoter.The sequence of target site is GCTGTGGAA (SEQ ID NO:1), and the location as shown in Figure 1.This proteinic sequence is as follows, and the line part is that zinc refers to cog region:

KKKQHICHIQGCGKVYG QRSNLVRHLRWHTGERPFMCTWSYCGKRFT RSD ALSRHKRTHTGEKKFACPECPKRFM QSSDLRRHIKTHQNK(SEQ?ID?NO：2)。

Obtaining with the double cross selective system can be in conjunction with other Zinc finger domain of SEQ ID NO:1.Referring to (2000) Proc.Natl.Acad.Sci.USA 97:7382-7287 such as for example U.S. Patent Application Publication No. 2003/0044787 (on March 6th, 2003) and Joung.Its aminoacid sequence is seen Fig. 6.

With the nucleotide sequence of these zinc finger proteins of coding be blended in coding VP16 activation domain, nuclear localization signal (NLS) and FLAG epi-position label nucleotide sequence, to produce engineered transcription factor.Exemplary polynucleotide sequence is seen Fig. 3 and 5, and its encoding amino acid sequence is seen Fig. 2 and 4 respectively.

Embodiment 3: the expression that improves the immunoglobulin gene of SR α promoters driven by engineered zinc finger protein

The DG44CHO cell line that contains the integration antibody expression construction of SR α promoters driven with 20-250ng coding VP16 activation domain (NVF) or ZFP 2392/00 (SEQ ID NO:2) the plasmid transient transfection (2392-VP16) that is connected in VP16.With PCR in real time (Taqman ^) mensuration immunoglobulin kappa chain mRNA expression.The κ chain mRNA that presentation of results shown in Figure 7,2392/00 transcription factor drive SR α transcribes has increased 4-5 doubly.

In measuring of the experiment of 2392/00-VP16 fusions to the effect of protein level, with coding VP16 activation domain (NVF) or be connected in two kinds of cell-derived cell lines of different DG44CHO of plasmid transient transfection (cell line A and cell line B) of the ZFP 2392/00 (2392) of VP16 activation domain, these two kinds of cell line stably express are expressed gamma heavy chain and the κ light chain by SR α promoters driven.Measure the expression of secreted immunoglobulin G with ELISA.Presentation of results shown in Figure 8, in two kinds of cell lines, the 2392/00ZFP-VP16 fusions has increased the IgG expression.

Whether can in the optimization expression system, increase expression in order to measure the 2392/00-VP16 fusion rotein, adopt the cell line that contains amplification gamma heavy chain and κ light chain cdna of selecting acquisition with methotrexate.The sequence that coding 2392/00-VP16 is merged ZFP is introduced the cell (ZFP) of this " high yield " cell line, with the mRNA expression with do not exist the same cell (NT) of ZFP to compare.Fig. 9 demonstrates, and when 2392/00-VP16 albumen is introduced these cells, can make the optimum level of γ chain and κ chain mRNA improve three times again.

Contain all cell line of the integration cDNA of the immunoglobulin G gamma heavy chain under SR α promoter transcription is controlled and κ light chain parts of coding with the plasmid transfection of coding 2392/00-VP16 fusion rotein.Selection has stablizes the transfectional cell that ZFP expresses.With the cloned cell line excretory IgG of ELISA mensuration from stably express 2392/00-VP16 fusion rotein.Result shown in Figure 10 discloses, and compares with the contrast non-transfected cells, increases about 4 times with IgG secretion in the protein stabilized cells transfected of 2392/00-VP16.

Embodiment 4: the selection and the characteristic of stable cell lines that contains the integration sequence of coding 2392/10-VP16 fusion rotein

With Chinese hamster ovary (CHO) the DG44 cell of plasmid transfection adherent growth on 6 orifice plates of coding 2392/10-VP16 fusion rotein, as described below.In 1ml serum-free growth medium, cell was hatched 4 hours with 1 μ g DNA and 4 μ lLipofectamine 2000, absorb culture medium then, add the conventional growth medium of 2ml.After 3 days, with various dilution factors cell is divided and to carry out zero mycin in the 25cm culture dish and select.When selection was finished, the single colony of results was transferred in 24 orifice plates and is cultivated from suitable dilution plate.The standard that selection is finished is the individual cells colony to occur and do not have new cell death, and it is all dead to observe the untransfected DG44 cell of putting under the selection pressure simultaneously.Analyze ZFP expression, the cell growth characteristics of 22 cloned cell lines and contained the activation of the report construction of multimerization ZFP binding site.

With one of these cell lines sub-clone, and from two sub-clones, extract genomic DNA.With BamHI restriction endonuclease catapepsis DNA, on the 0.9%TAE agarose gel, separate, transfer to Nytran ⁺On the film, detect this film with the 290bp radioactive mark DNA fragment of coding VP16 activation domain.Detect the single band of about 12kbp in two kinds of cell lines that contain ZFP, there is the fusion rotein coding DNA of a copy in this explanation.In the DNA of wild type DG44 Chinese hamster ovary celI, do not detect this band.

Embodiment 5: the SR α promoter that makes up the target site of the ZFP-VP16 fusion rotein that contains multicopy

The dna fragmentation (SEQ ID NO:1) that will contain 6 2392/00 and 2392/10ZFP target site copy is connected in the dna fragmentation that contains SR α promoter is called " SR α Z6 " with structure promoter.Therefore, the Z6 promoter contains 7 SEQ ID NO:1 copies.The activation level that the ZFP that these extra target sites produce this promoter mediates is much higher.As described below.

Insert the insert that contains extra 6 binding sites after at first those restriction sites (following represent with runic) being inserted the SV40 part upstream of SR α promoter, become the BamHI/NdeI fragment.Underscore partly is 7 repetitions of target site.The target site in downstream, NdeI site is the site that the SV40 part Central Plains of SR α promoter pre-exists.

The SR α Z6 promoter sequence partly that contains the ZFP target site is as follows:

BamHI

ggatccga gctgtggaatgaga gctgtggaatgaga gctgtggaatgaga gctgtggaatgaga gctgtggaatgaga gctgtggaatgacatatg gctgtggaatgtgtgtcagtta NdeI

(SEQ?ID?NO：23)

Embodiment 6: make up the CMV promoter that contains a plurality of ZFP-VP16 fusion rotein target site copies

Make up the promoter that is called " CMVz10 " by a dna fragmentation (SEQ ID NO:1) is inserted the MluI restriction site that is positioned at CMV promoter upstream just, this dna fragmentation comprises the copy of a plurality of 2392/00 and 2392/10 engineered ZFP target site.The perfection that the CMVz10 promoter contains 9 target sites repeats, and the tenth is repeated is 8/9 coupling (C-T replaces).The CMVz10 promoter sequence is wherein represented the MluI restriction site with runic as follows, and underscore partly is a target site.It is noted that owing to oppositely insert this sequence, shown in serial response the complement of each binding site.

MluI

acgcgttca ttccacagctctca ttccacagctctca ttccacagctctca ttccacagctctca ttccacagctctca ttcca cagctctca ttccacagctctca ttccacagctctca ttccacagctctca ttccacagtcacgcgt MluI

(SEQ?ID?NO：24)

Also make up with having detected and comprise the binding site different and other promoter construction of different binding sites with promoter orientation with promoter quantity.

Embodiment 7: improve protein output by the SR α promoter that contains a plurality of ZFP-VP16 fusion rotein target site copies

Clone and separate thing (2392/10-7) (embodiment 4) and parent DG44 cell that the DG44 Chinese hamster ovary celI that contains 2392/10 ZFP of stable integration with two kinds of different antibody expression construction transfections is.In an example, under the control of each comfortable SR α promoter (SRa) of heavy chain and light chain transcript unit; In another example, heavy chain and light chain transcript unit are all under the control that contains just in the SR α promoter (SRa 2393BS) of extra 8 ZFP target sites in promoter downstream.After the transfection 3 days, measure the IgG secretion with ELISA.

The results are shown in Figure 11, this result shows the IgG expression depends on whether there is 2392/10ZFP, than the higher IgG level of 2392/10 target site generation of multicopy.

Embodiment 8: strengthened by the CMV promoter that contains a plurality of ZFP-VP16 fusion rotein target site copies and transcribe

In order to detect the 2392/10-VP16 fusion rotein, make up the report construction that CMV promoter operability is connected in green fluorescent protein (GFP) coded sequence to the active influence of CMV promoter transcription.Make up the variant of this report construction then, it contains the copy of a plurality of 2392/10 ZFP target sites, and this target site is in core CMV promoter sequence upstream.Detect the promoter of the target site of the varying number that contains different orientation by the following method: they are transfected into 2392/10-7 cell line (embodiment 4), use PCR in real time (Taqman then ^) analysis GFP mRNA level.Representative result is seen Figure 12.Presentation of results, than the higher steady-state mRNA level of target site generation of multicopy, the orientation of target site with respect to CMV promoter sequence remainder depended in this effect.In the parental cell line of not expressing 2392/10ZFP, do not observe the influence of the quantity or the orientation of target site.

Embodiment 9: increase erythropoietin by SR α that contains a plurality of 2392/10 target site copies and CMV promoter and express, this is fusion protein mediated by 2392/10-VP16.

Make up the construction of three kinds of different expression erythropoietin (Epo).In first kind, express (CMV) by the CMV promoter control Epo that is blended in the beta-globin intron.In second kind, express (CMVz10, embodiment 6) by the CMV promoter control Epo that contains ten upstream copies of 2392/10 binding site that is blended in the beta-globin intron.In the third, express (SR α Z6, embodiment 5) by the SR α promoter control Epo that contains six additional copies of 2392/10 binding site that are positioned at the promoter upstream just.Three kinds of construction transient transfections are gone into parent DG44 Chinese hamster ovary celI or expressed the 2392/10-7 cell line of ZFP.After 24 hours, measure the Epo secretion with ELISA.

Observed result before result shown in Figure 13 has confirmed, promptly when not having 2392/10-VP16ZFP, the CMV promoter is stronger than SR α promoter.Yet 2393/10-VP16 albumen activates SR α Z6 promoter can improve its activity, so that it is suitable with the CMV promoter.And the adjacent position of 2392/10 target site being inserted the CMV promoter in the cell of expressing 2392/10-VP16 can make its stronger activity increase by three times again.Therefore, adopt engineered ZFP transcriptional activator to improve the activity of the strongest natural generation promoter of known activity.

By with reference to all patents as herein described, patent application with deliver thing and include this paper in full in.

Though provide the disclosure for the purpose of setting forth and understanding in detail by the mode that illustrates and give an example, it will be understood by those skilled in the art that and under the situation that does not deviate from design of the present invention or scope, to carry out various changes and modification.Therefore, should not think that above-mentioned description and embodiment are restrictive.

Claims (18)

1. regulate the method that nucleotide sequence is transcribed for one kind in cell, described method comprises:

Express the protein that comprises SEQ ID NO:7 in cell, wherein said protein bound is in target site;

Wherein said target site operability is connected in described nucleotide sequence.

2. the method for claim 1 is characterized in that, described target site comprises SEQ ID NO:1.

3. method as claimed in claim 2 is characterized in that, a plurality of target site operability are connected in described nucleotide sequence.

4. the method for claim 1 is characterized in that, described nucleotide sequence comprises the cDNA sequence.

5. the method for claim 1 is characterized in that, described protein also comprises transcription activating domain.

6. method as claimed in claim 5 is characterized in that, described transcription activating domain is the VP16 domain.

7. regulate the method that first and second nucleotide sequences are transcribed for one kind in cell, described method comprises:

Express the protein that comprises SEQ ID NO:7 in cell, wherein said protein bound is in target site;

Wherein, described target site operability is connected in described first and second nucleotide sequences.

8. method as claimed in claim 7 is characterized in that, described target site comprises SEQ ID NO:1.

9. method as claimed in claim 8 is characterized in that, a plurality of target site operability are connected in described first nucleotide sequence.

10. method as claimed in claim 8 is characterized in that, a plurality of target site operability are connected in described second nucleotide sequence.

11. method as claimed in claim 8 is characterized in that, a plurality of target site operability are connected in described first and second nucleotide sequences.

12. method as claimed in claim 7 is characterized in that, described first nucleotide sequence comprises the cDNA sequence.

13. method as claimed in claim 7 is characterized in that, described second nucleotide sequence comprises the cDNA sequence.

14. method as claimed in claim 7 is characterized in that, described first and second nucleotide sequences all comprise the cDNA sequence.

15. method as claimed in claim 14 is characterized in that, described cDNA sequential coding antibody polypeptides.

16. method as claimed in claim 15 is characterized in that, a described cDNA sequential coding heavy chain of antibody, described the 2nd cDNA sequential coding light chain of antibody.

17. method as claimed in claim 7 is characterized in that, described protein also comprises transcription activating domain.

18. method as claimed in claim 17 is characterized in that, described transcription activating domain is the VP16 domain.

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