WO2001049832A2 - Transduction de recombinases pour ciblage genetique inductible - Google Patents

Transduction de recombinases pour ciblage genetique inductible Download PDF

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WO2001049832A2
WO2001049832A2 PCT/EP2001/000060 EP0100060W WO0149832A2 WO 2001049832 A2 WO2001049832 A2 WO 2001049832A2 EP 0100060 W EP0100060 W EP 0100060W WO 0149832 A2 WO0149832 A2 WO 0149832A2
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gly
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PCT/EP2001/000060
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WO2001049832A3 (fr
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Frieder Schwenk
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Artemis Pharmaceuticals Gmbh
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Priority claimed from EP00100351A external-priority patent/EP1118668A1/fr
Application filed by Artemis Pharmaceuticals Gmbh filed Critical Artemis Pharmaceuticals Gmbh
Priority to CA002396149A priority Critical patent/CA2396149A1/fr
Priority to AU33683/01A priority patent/AU3368301A/en
Priority to JP2001550361A priority patent/JP2003518947A/ja
Priority to IL15051001A priority patent/IL150510A0/xx
Priority to EP01905646A priority patent/EP1244796A2/fr
Publication of WO2001049832A2 publication Critical patent/WO2001049832A2/fr
Publication of WO2001049832A3 publication Critical patent/WO2001049832A3/fr

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention provides the use of a fusion protein comprising a site-specific DNA recombinase domain and a protein transduction domain for preparing an agent for inducing target gene alteration in a living organism or in cultured cells, suitable fusion proteins and a method for the production of said fusion proteins.
  • ES cells totipotent mouse embryonic stem (ES) cells
  • Capecchi Trends in Genetics 5, 70 - 76 (1989)
  • ES cells can pass mutations induced in vitro to transgenic offspring in vivo, it is possible to analyze the consequences of gene disruption in the context of the entire organism.
  • numerous mouse strains with functionally inactivated genes (“knock out mice”) have been created by this technology and utilized to study the biological function of a variety of genes.
  • conditional mutagenesis employs a site-specific recombination system (e.g. Cre/loxP or Flp/frt - Sauer and Henderson, N. Proc. Natl. Acad. Sci. USA 85, 5166- 5170 (1988); Senecoff et al., 3. Mol. Biol., 201, 405 - 421 (1988)) which enables a temporally and/or spatially restricted alteration of target genes (Rajewsky et al., J. Clin. Invest., 98, 600 - 603 (1996)).
  • the creation of conditional mouse mutants requires the generation of two mouse strains, i.e.
  • the recombinase recognition strain is generated by homologous recombination in ES cells as described above except that the targeted exon(s) is (are) flanked by two recombinase recognition sequences (hereinafter "RRS"; e.g. loxP or frt).
  • RRS recombinase recognition sequences
  • the type of recombination event mediated by the recombinase depends on the disposition of the RRS, with deletions, inversions, translocations and integrations being possible (Torres and K ⁇ hn, Oxford University Press, Oxford, New York (1997)). By placing the RRS into introns, an interference with gene expression before recombination can be avoided.
  • the recombinase expressing strain contains a recombinase transgene (e.g. Cre, Flp) whose expression is either restricted to certain cells and tissues or is inducible by external agents.
  • Crossing of the recombinase recognition strain with the recombinase expressing strain recombines the RRS-flanked exons from the doubly transgenic offspring in a prespecified temporally and/or spatially restricted manner.
  • the method allows the temporal analysis of gene function in particular cells and tissues of otherwise widely expressed genes. Moreover, it enables the analysis of gene function in the adult organism by circumventing embryonic lethality which is frequently the consequence of gene mutation.
  • inducible mutations provide an excellent genetic tool.
  • the current systems for inducible recombinase expression in transgenic animals suffer from a certain degree of leakiness in the absence of the inducer (K ⁇ hn et al., Science 269(5229): 1427-9 (1995); Schwenk et al., Nucleic Acids Res.; 26(6): 1427-32 (1998)).
  • conditional mutants is a time consuming and labor intensive procedure, since the recombinase recognition strain and the recombinase expressing strain have to be breed at least over two generations in order to obtain animals carrying both, the recombinase transgene and two copies of the RRS-flanked target gene sequence.
  • PTD Protein tranduction domains
  • WO 99/29721 moreover mentions TAT mutants having an enhanced activity as compared to the wild-type peptide. Fusion of PTDs to heterologuous proteins conferred the ability to transduce into cultured cells (Fawell et al., Proc. Natl. Acad. Sci.
  • WO 99/11809 mentions a fusion protein Antp-Cre and emphasizes that it may be used to deliver the Cre into the cell which recombines inside the cell nucleus. It is mentioned that the fusion protein is suitable for manipulating genomic DNA at precise locations in a temporal regulated manner.
  • WO 99/60142 discloses vector constructs for gene therapy carrying a tumor cell sensitizing gene, a sensitizing gene expression regulatory system, a control gene and a control gene expression regulatory system, wherein the control gene can be a fusion gene consisting of a recombinase (viz. Cre or Flp) and a trafficking protein (viz. VP22).
  • the Antennapedia PTD is not a generally applicable transducing protein, namely it has only a limited activity with proteins having more than 100 amino acid residues (Derossi et al., Trends Cell Biol. 8: 84-87, 1998).
  • the limited transducing activity of the Antp PTD and the size of the generally known recombinases ranging from about 200 to about 600 amino acid residues
  • site-specific DNA recombinase proteins can be translocated into cells of a living organism when fused to specific protein transduction domains, namely transduction domains being derived from the VP22 protein of HSV or from the TAT protein of HIV.
  • transduction domains being derived from the VP22 protein of HSV or from the TAT protein of HIV.
  • the present invention thus provides
  • a protein transduction domain for preparing an agent for inducing target gene alterations in a living organism or cell culture, wherein said living organism carries at least one or more recognition sites for said site-specific DNA recombinase integrated in its genome;
  • (2) a method for inducing gene alterations in a living organism which comprises administering to said living organism a fusion protein comprising a site-specific DNA recombinase domain and a PTD as defined in (1) above, wherein said living organism carries at least one or more recognition sites for said site-specific DNA recombinase integrated in its genome;
  • a PTD being derived from the VP22 protein of HSV or from the TAT protein of HIV provided that when the site-specific DNA recombinase domain is wild-type Cre or Flp then the PTD is not the full length VP22 PTD of HSV (i.e., the fusion protein is not identical to the fusion protein of Dalby and Bennett, Invitrogen, Expressions 6.2, page 13 (1999) and of WO 99/60142);
  • a method for producing the fusion protein of (1) above which comprises culturing the transformed host cell of (6) above and isolating the fusion protein;
  • Fig. 1 Generation of induced mouse mutants using purified fusion proteins.
  • A Expression of the fusion protein consisting of the site-specific DNA recombinase (e.g. Cre) and the protein transduction domain (e.g. the HIV derived TAT peptide) in prokaryotic or eukaryotic cells.
  • site-specific DNA recombinase e.g. Cre
  • protein transduction domain e.g. the HIV derived TAT peptide
  • B Extraction and purification of the expressed fusion protein (e.g. as described in Nagahara et al., Nat. Med. 4 (12): 1449-52 (1998)).
  • C Injection of the purified fusion protein into mice carrying the RRS- flanked target sequence.
  • Fig. 2 Scheme of the bacterial expression vector pT7-TACS (SEQ ID NO: 16).
  • the coding region of the 11 amino acid protein transduction domain of HIV TAT protein is fused to the N-terminus of the Cre recombinase protein sequence.
  • the 10-amino-acid strep tag and the protease factor Xa recognition sequence are fused to the C-terminus.
  • the T7 promoter permits expression of TAT-Cre protein in E. coli.
  • Fig. 3 Detection of purified TAT-Cre protein by Coomassie staining and Western blot analysis.
  • A Coomassie stained SDS-PAGE gel. Lane 1: 10 kDa ladder (Life Technologies, Cat. No.: 10064-012), 2: 1000 ng BSA, 3: 750 ng BSA, 4: 500 ng BSA, 5: 100 ng BSA, 6: 50 ng BSA, 7: 5 ⁇ l TAT-Cre, 8: 1 ⁇ l TAT- Cre in Bicine buffer.
  • Lane 1 Western blot analysis using an alkaline phosphatase-conjugated anti- strep tag antibody (IBA, Cat. No: 2-1503-001).
  • Lane 1 MultiMark (Invitrogen, Cat. No.: LC5725), 2: 7 ⁇ l TAT-Cre, 3: 5 ⁇ l TAT-Cre, 4: 2,5 ⁇ l TAT-Cre, 5: 1,25 ⁇ l TAT-Cre in Bicine buffer.
  • Fig. 4 X-Gal staining of M5Pax8 cells treated with TAT-Cre protein.
  • M5Pax8 fibroblasts where treated for 18 h with 3,5 (A), 6,9 (B) and 13,8 ⁇ g/ml TAT-Cre protein (C) in serum-free medium.
  • C TAT-Cre protein
  • Fig. 5 Measurement of ⁇ -galactosidase activity in cell lysates.
  • M5Pax8 fibroblasts where treated for 18 h with increasing concentrations of TAT- Cre, as indicated, or transiently transfected with either expression vectors for Cre (pCMV-I-Cre-pA, see SEQ ID NO: 29) or ⁇ -galactosidase (pCMV-I- ⁇ -pA, see SEQ ID NO:30).
  • Cre pCMV-I-Cre-pA
  • pCMV-I- ⁇ -pA see SEQ ID NO:30
  • Fig. 6 PCR detection of TAT-Cre mediated recombination in mice.
  • A PCR-analysis of genomic DNA from duodenum (lane 2), liver (3), kidney (4), spleen (5), muscle (6), lung (7), tail (8) and brain (9) of a plnl3 mouse treated three times with intraperitoneal injections of 75 ⁇ g TAT Cre protein at two-day-intervals. Deletion of the loxP-flanked DNA segment is indicated by the presence of the about 400 bp fragment.
  • Lane 1 1-kb-ladder (Life Technologies).
  • B PCR strategy to detect Cre-mediated deletion of the loxP-flanked DNA segment. Arrows indicate the positions of the primers.
  • C PCR-analysis of genomic DNA from spleen of a plnl3 mouse treated three times with intraperitoneal injections of 75 ⁇ g TAT Cre protein at two- day-intervals (lane 4). To confirm the presence of the BamH I restriction site, the PCR product was digested with BamH I which produces two diagnostic fragments of about 190 and about 210 bp (5). As a control, tail DNA from untreated mice carrying the loxP-flanked (lane 2) and the detected plnl3 allele (3) was subjected to PCR amplification. Lane 1: 100 bp ladder (Life Technologies), lane 6: 1 kb ladder (Life Technologies).
  • Fig. 7 Scheme of the bacterial expression vectors pT7-VPCS (SEQ ID NO: 17) and pCRT7- ⁇ VPCS (SEQ ID NO: 15).
  • the coding region of the 301 amino acid protein transduction domain of HSV VP22 protein (A) or the truncated 143 amino acid ⁇ VP22 domain (B) is fused to the N-terminus of the Cre recombinase protein sequence.
  • the 10-amino-acid strep tag and the protease factor Xa recognition sequence are fused to the C-terminus.
  • the T7 promoter allows the expression of VP22-Cre and ⁇ VP22-Cre fusion proteins in E. coli.
  • the sequence in pCRT7- ⁇ VPCS encoding the 15 amino acid N-terminal leader sequence is used for enhanced protein stability (Invitrogen).
  • Fig. 8 Detection ,of the purified VP22-Cre and ⁇ VP22-Cre fusion proteins by Coomassie staining and Western blot analysis.
  • A Detection of VP22-Cre protein in a Coomassie-stained SDS-PAGE gel. Lane 1: 10 kDa ladder, 2: 1000 ng BSA, 3: 500 ng BSA, 4: 100 ng BSA, 5: inclusion body protein extract before chromatography, 6: unbound protein, 7: fraction 17, 8: fraction 18, 9: fraction 19, 10: fraction 20.
  • the position of the 75 kDa VP22-Cre protein is indicated by the arrow head.
  • Lane 1 MultiMark (Invitrogen), 2: inclusion body protein extract before chromatography, 3: unbound protein, 4: fraction 10, 5: fraction 11, 5: fraction 16, 6: fraction 17, 7: fraction 18, 8: fraction 19, 9: fraction 19, 10: fraction 20.
  • Fig. 9 X-Gal staining of M5Pax8 cells treated with VP22-Cre and ⁇ VP22- Cre fusion proteins.
  • M5Pax8 fibroblasts where treated for 18 h with either Bicine buffer (A), 0.5 ⁇ g/ml VP22-Cre (B) or 3.75 g/ml ⁇ VP22-Cre (C) in serum-free medium.
  • A Bicine buffer
  • B 0.5 ⁇ g/ml VP22-Cre
  • C 3.75 g/ml ⁇ VP22-Cre
  • Fig. 10 Measurement of ⁇ -galactosidase activity in cell lysates.
  • M5Pax8 fibroblasts where treated for 18 h with VP22-Cre, ⁇ VP22-Cre or Bicine buffer alone, as indicated or transiently transfected with expression vectors for Cre (pCMV-I-Cre-pA, see SEQ ID NO: 29) or ⁇ -galactosidase (pCMV-I- ⁇ -pA, see SEQ ID NO:30).
  • Cre pCMV-I-Cre-pA
  • pCMV-I- ⁇ -pA see SEQ ID NO:30
  • Fig. 11 PCR detection of Cre mediated recombination in cells treated with VP22-Cre and ⁇ VP22-Cre fusion proteins shown in SEQ ID NOs: 21 and 14, respectively).
  • A PCR-analysis of genomic DNA isolated from M5Pax8 fibroblasts.
  • Cells were transiently transfected with a Cre expression vector (lane 2) or treated for 18 h with either buffer alone (lane 3), 7.5 ⁇ g/ml VP22-Cre (4, 5) or 15 ⁇ g/ml ⁇ VP22-Cre (6, 7) in serum-free medium.
  • a Cre expression vector lane 2
  • 7.5 ⁇ g/ml VP22-Cre 4, 5
  • 15 ⁇ g/ml ⁇ VP22-Cre 6
  • the PCR products were digested with Nco I which produces two diagnostic fragments of 85bp and 141bp (lanes 5 and 7).
  • Lane 1 100 bp ladder (Life Technologies)
  • lane 8 1 kb ladder (Life Technologies).
  • B PCR strategy to detect Cre-mediated deletion of the loxP-flanked DNA segment. Arrows indicate the positions of the primers.
  • target sequences means all kind of sequences which may be mutated (viz. deleted, translocated, integrated and/or inverted) by the action of the recombinase.
  • the number of RRS in the target sequence depends on the kind of mutation to be performed by the recombinase. For most of the mutations (especially for deletions and invertions) two RRS are required which are flanking the sequence to be mutated (deleted or inverted). For some kinds of integrations only one RRS may be necessary within the target sequence.
  • the "living organisms" according to the present invention are multi-cell organisms and can be vertebrates such as mammals (e.g., rodents such as mice or rats) or non-mammals (e.g., fish) or can be invertebrates such as insects or worms, or can be plants (higher plants, algi or fungi). Most preferred living organisms are mice and fish.
  • Cell culture include cells isolated from the above defined living organism and cultured in vitro. These cells can be transformed (immortalized) or untransformed (directly derived from the living organism; primary cell culture).
  • the site-specific DNA recombinase domain within the fusion protein of the invention of the present application is preferably selected from a recombinase protein derived from Cre, Flp, ⁇ C31 recombinase (Thorpe and Smith, Proc. Natl. Acad. Sci, USA, vol. 95, 5505-5510 (1998)), ⁇ resolvase (Schwickardi and Dr ⁇ ge, FEBS letters 471:147-150 (2000) and R recombinase (Araki et al., 3. Mol. Biol., 182, 191-203 (1985)).
  • the preferred recombinases are Cre and mutants thereof (preferably the Cre variant of aa 15 to 357 of SEQ ID NO: 2 or aa 325-667 of SEQ ID NO: 6) and Flp and variants thereof including Flpe (preferably the Flp variant of aa 15 to 437 of SEQ ID NO: 4 or aa 325 to 747 of SEQ ID NO: 8).
  • the protein transduction domain according to the present invention includes, but is not limited to, the PTDs mentioned in Background of the
  • the PTD preferably is derived from the VP22 protein of HSV or from the TAT protein of HIV.
  • Suitable TAT proteins include, but are not limited to, proteins comprising (i) the amino acid sequence shown in SEQ
  • transduction domains consisting of the TAT proteins (i) and (ii) above.
  • Suitable VP22 proteins include, but are not limited to, the wild-type VP22 protein, i.e., a protein comprising amino acids 1 to 302 of SEQ ID No:21, and truncated forms thereof. Truncated VP22 proteins in accordance with the present invention can be those lacking 1 to 158 amino acid residues at their N-terminal end.
  • the most preferred VP22 protein is the truncated VP22 PTD comprising amino acid residues 16 to 157 of SEQ ID NO: 14.
  • the fusion of the two domains of the fusion protein can occur at any possible position, i.e., the protein transduction domain can be fused to the N- or C-terminal of the site-specific DNA recombinase or can be fused to active sites within the site-specific DNA recombinase.
  • the protein transfusion domain is fused to the N-terminal of the site-specific DNA recombinase domain.
  • the protein transduction domain can be fused to the site-specific DNA recombinase either through a direct chemical bond or through a linker molecule.
  • Such linker molecule can be any bivalent chemical structure capable of linking the two domains.
  • the preferred linker molecule according to the present invention is a short peptide, e.g., having 1 to 20, preferably 1 to 10, amino acid residues.
  • Specifically preferred short peptides are essentially consisting of Gly, Ala and/or Leu.
  • the fusion protein of the invention of the present application may further comprise other functional sequences such as secretion conferring signals, nuclear localisation signals and/or signals conferring protein stabilisation.
  • the DNA sequence coding for said fusion protein preferably comprises the sequence 5' TAG GGC CGC AAG AAG CGC CGC CAA CGC CGC CGC 3'.
  • Such a preferred DNA sequence is for instance shown in SEQ ID NO: 11.
  • the 3" terminal codon ggc codes for the linker Gly.
  • the DNA sequence of a suitable recombinase may be directly attached to said codon ggc.
  • the fusion protein can be obtained by the following steps:
  • TAT-mediated delivery of active Cre protein works with sufficient efficacy to facilitate inducible gene targeting both in cell lines and living organisms.
  • a vector for the expression of a TAT-Cre fusion protein in E. coli was constructed, TAT-Cre protein was expressed in E. coli and purified from bacterial lysates.
  • a reporter cell line that contains a loxP-containing reporter construct was used. This reporter, when recombined by Cre recombinase, allows the expression of a ⁇ - galacosidase gene.
  • a transgenic mouse strain carrying a loxP- flanked target was used to invest the activity of the TAT-Cre protein in " vivo.
  • VP22-mediated delivery of active Cre protein works with sufficient efficacy to facilitate inducible gene targeting.
  • Bacterial expression vectors were constructed for the production of VP22-Cre fusion proteins in E. coli. The activity of purified VP22-Cre proteins were tested using a reporter fibroblast cell line containing a loxP-flanked reporter construct.
  • the injection of the purified fusion protein of the present invention into a living organism e.g., a mouse
  • a gene comprising the RRS-flanked target sequence e.g., in an amount of 1 to 200, preferably 5 to 50 ⁇ g per g body weight.
  • a reporter mouse strain carrying an RRS-flanked cassette was used (Thorey et al., Mol. Cell Biol., 18(10):6164 (1998)).
  • Analysis is achieved by determining the pattern of induced target gene recombination (e.g. through PCR analysis, Southern blot analysis or X-Gal staining on tissue sections; Maniatis et al., 1989; Gossler and Zachgo, Joyner AL (Ed.), Oxford University Press, Oxford, New York (1993)).
  • a reporter cell line containing a loxP-containing reporter construct was used to test the activity of the TAT-Cre protein in vitro. This reporter, when recombined by Cre recombinase, allows the expression of a ⁇ - galacosidase gene.
  • TAT-Cre coding region was generated by PCR using Advantage-HF PCR Kit (Clontech), 20 pmol of the primers TATcre sense (5'-atg cca tgg get acg gcc gca aga age gcc gcc aac gcc gcc gcg gca tgt cca att tac tga ccg tac acc-3'; SEQ ID NO: 31) and TATcre antisense (5'-ttt egg ate cgc cgc ata ace agt g-3'; SEQ ID NO: 32) and 10 ng pCMV-I-Cre-pA (see SEQ ID NO: 29) as template.
  • the PCR reaction was performed using the following cycle profile: 2' 94 °C, 4 x (30" 94 °C min, 30" 50 °C, 1' 72 °C), 12 x (30" 94 °C min, 30" 55 °C, 1' 72 °C) and 10' 72 °C.
  • the resulting PCR fragment was digested with Nco I and BamH I, treated with Klenow enzyme and ligated into the plasmid pBSII KS+ which had been opened with restriction enzyme BamH I, treated with Klenow and dephosphorylated with calf intestinal phosphatase.
  • the resulting plasmid pBS TAT-5'cre was verified by DNA sequencing.
  • Plasmid pCMV-I-Cre-pA (SEQ ID NO: 29) was digested with Age I and Sal I which released a 1,036 kb fragment containing the 3' part of the Cre coding region. This fragment was ligated into the plasmid pBS TAT-5'cre which had been opened with Age I and Sal I.
  • the following cycle profile was used: 2' 94 °C, 25 x (30" 94 °C min, 15" 54,6 °C, 2'30" 68 °C).
  • the amplified PCR fragment was purified using GFX columns (Amersham Pharmacia), digested with Xba I and ligated into the plasmid pASK57 (Skerra and Arne, Gene 151: 131-135 (1994)) which had been opened with restriction enzymes Xba I and Eco 47 III and dephosphorylated with calf intestinal phosphatase.
  • the resulting plasmid pASK75-TACS was digested with restriction enzymes Nco I and Hind III which released a 1,1 kb fragment.
  • the fragment was subsequently ligated into the plasmid pT7- 7 (Studier and Moffatt, J. Mol. Biol. 189: 113-130 (1986)) which had been opened with restriction enzymes Nco I and Hind III and dephosphorylated with calf intestinal phosphatase resulting in the plasmid pT7-TACS (SEQ ID NO: 16).
  • PT7-VPCS The Cre coding region was generated by PCR using Advantage-HF PCR Kit (Clontech), 20 pmol of the primers VP22cre sense (5'-taa eta gcg gcc gca tgt cca att tac tga ccg tac ac-3'; SEQ ID NO: 35) and VP22cre antisense (5'-tcg age ggc cgc cat cgc cat ctt cca gca ggc g-3'; SEQ ID NO:36) and 10 ng pgkcre-pA (SEQ ID NO:40) as template.
  • the PCR reaction was performed using the following cycle profile: 2' 94 °C, 5 x (30" 94 °C, 30" 50 °C, 2' 72 °C), 15 x (30" 94 °C, 30" 55 °C, 2' 72 °C) and 10' 72 °C.
  • the resulting PCR fragment was digested with Not I and ligated into the plasmid pVP22/Myc-His (Invitrogen), which had been opened with restriction enzyme Notl, dephosphorylated with calf intestinal phosphatase.
  • the resulting plasmid pVP22-cre myc/His was verified by DNA sequencing.
  • the amplified PCR fragment was purified using GFX columns (Amersham Pharmacia), digested with Xba I and ligated into the plasmid pASK57 (Skerra and Arne, Gene 151: 131-135 (1994)) which had been opened with restriction enzymes Xba I and Eco 47 III and dephosphorylated with calf intestinal phosphatase.
  • the resulting plasmid pASK75-VPCS was digested with restriction enzymes Nde I and Hind III which released a 2,0 kb fragment. The fragment was subsequently ligated into the plasmid pT7-7 (Studier and Moffatt, 3. Mol. Biol.
  • PCRT7- ⁇ VPCS The ⁇ VP22-Cre coding region was generated by PCR using Platinum Pfx DNA polymerase (Life Technologies), 20 pmol of the primers FPA007 (5'-ttc cga aga cga cga aac acc-3"; SEQ ID NO:38) and FPA008 (5'-tat att cga age tta tta ace ace gaa ctg cg-3'; SEQ ID NO:39) and 30 ng pT7-VPCS (SEQ ID NO: 17) as template.
  • the PCR reaction was performed using the following cycle profile: 2' 94 °C, 25 x (30" 94 °C, 30" 61 °C, 2'30" 68 °C) and 7' 68 °C.
  • the resulting 1,8 kb PCR fragment was digested with Nco I and Sfu I and ligated into the plasmid pCRT7/VP22-l (Invitrogen), which had been opened with restriction enzymes Nco I and Sfu I, and dephosphorylated with calf intestinal phosphatase.
  • the resulting plasmid pCRT7- ⁇ VPCS (SEQ ID NO: 15) was verified by DNA sequencing. Expression of the fusion proteins in E. coli: E.
  • coli BL21(DE3)-RIL cells (Stratagene) were transformed with pT7-TACS and grown on LB agar plates containing .100 ⁇ g/ml ampicillin.
  • E. coli BL21(DE3)-RP cells (Stratagene) were transformed with pT7-VPCS and grown on LB agar plates containing 100 ⁇ g/ml ampicillin.
  • E. coli BL21(DE3)-pLysS (Invitrogen) were transformed with pCRT7- ⁇ VPCS and grown on LB agar plates containing 25 ⁇ g/ml kanamycine and 34 ⁇ g/ml chloramphenicol. Single colonies were isolated and used to prepare glycerol stocks.
  • Protein extract was centrifuged at 31000xg and supernatant harvested. Protein extract was diluted in an equal volume of Chromatography buffer A (50mM Bicine, pH 8,5). PH was adjusted to pH 8,5 and the extract was filtered through a 0,45 ⁇ m filter (Millipore).
  • FPLC Akta Explorer, Amersham Pharmacia
  • a cation exchange column Sepharose SP, Column body HR_5/5 (0.5 x 5cm), column volume (CV) 1ml, linear flow 300cm/hour, Amersham Pharmacia). After addition of sample to FPLC column, buffer was exchanged with Chromatography buffer A at 10 CV.
  • TAT-Cre and VP22-Cre fusion proteins were eluted from the column by gradient elution using chromatography buffer B (50mM Bicine, 1M NaCl, pH 8,5) using the following profile: 0 - 50 % buffer B, 0 CV; 50 % buffer B, 10 CV; 50 - 100 % buffer B (linear gradient), 20 CV; 100 % buffer B, 10 CV.
  • chromatography buffer B 50mM Bicine, 1M NaCl, pH 8,5
  • ⁇ VP22-Cre protein was eluted from the column by gradient elution using the following profile: 0 - 10 % buffer B, 0 CV; 10 % buffer B, 10 CV; 10 - 30 % buffer B, 0 CV; 30 % buffer B, 10 CV; 30 - 100 % buffer B, 0 CV; 100 % buffer B, 10 CV.
  • Three 1,5ml fractions each containing purified fusion proteins were collected. Purity and concentration of protein fractions were determined by Coomassie blue stained SDS-PAGE gels and Western blot analysis using dilutions of BSA standard solutions. In addition protein content was determined using a Bradford assay (Coomassie Plus protein assay, Pierce).
  • SDS-PAGE and Western blot analysis SDS-PAGE and Coomassie staining was performed according to standard protocols (Maniatis et al., Cold Spring Harbor Laboratory, New York (1989)) using 4 - 12 % gradient SDS-polyacrylamide gels (NuPAGE, Invitrogen, cat. no. : NP0321).
  • Western blot analysis was performed using a Semi-Try Blotting Chamber (Biorad) and nitrocellulose membranes (0,2 ⁇ m; Schleicher & Schuell) according to the manufacturers protocols.
  • the fusion proteins were detected by using an alkaline phosphatase-conjugated anti-strep tag antibody (IBA, Cat. No.: 2-1503-001) according to the manufacturers protocol.
  • M5Pax8 Cre reporter cell line The SV40-transformed murine embryonic fibroblast line MEF5/5 (Schwenk et al., Nucl Acids Res 26(6), 1427-32 (1998)) was transfected with the vector pPGKpaXl (Kellendonk et al, Nucl. Acids Res. 24, 1404-11 (1996)). 10 6 MEF5/5 cells were electroporated with 20 ⁇ g pPGKpaXl plasmid DNA linearised with Sea I and plated into 48-well-plates. The cells were cultured in DMEM/Glutamax medium (Life Technologies) supplemented with 10 % fetal calf serum at 37°C, 10 % C0 2 in humid atmosphere.
  • DMEM/Glutamax medium Life Technologies
  • ⁇ -galactosidase activity Fibroblasts (10 6 cells per 24 well plate (Falcon)) were transfected with 25 ng pCMV-I- Cre-pA (see SEQ ID NO:29) or pCMV-I- ⁇ -pA (see SEQ ID NO:30) plasmids using the FuGene transfection reagent (Roche Diagnostics). After 2 days the cells were lysed and the ⁇ -galactosidase activities were determined with the ⁇ -galactosidase reporter gene assay (Roche. Diagnostics) according to the manufacturers guidelines using a Lumistar luminometer (MWG).
  • MWG Lumistar luminometer
  • fibroblast cells were washed once with phosphate buffered saline (PBS), and the cells were fixed for 5 minutes at room temperature in a solution of 4% formaldehyde in PBS. Next, the cells were washed twice with PBS and finally incubated in staining solution for 24 hours at 37°C (staining solution: 5 mM K3(Fe(CN)6), 5mM K4(Fe(CN)6), 2mM MgCI2, lmg/ml X-Gal (BioMol) in PBS). Blue stained, ⁇ -galactosidase positive cells were detected and distinguished from negative (transparent) cells in a cell culture binocular microscope under 200x magnification. For each determination a minimum of 200 cells was counted.
  • PBS phosphate buffered saline
  • PCR detection of Cre-mediated recombination Genomic DNA extracted from tissue samples was subjected to PCR using Taq-polymerase (Gibco BRL Cat. No. 10342-020) using 20 pmol of each primer (sense: 5' -CAT CTC CGG GCC TTT CGA CCT G - 3', antisense: 5' -GCG ATC GGT GCG GGC CTC TTC - 3'; SEQ ID Nos: 41 and 42, respectively). PCR was performed using the following cycle profile: 2' 94°C, 35 x (30" 94°C, 30" 55 °C, 1' 72 °C), 10 min 72 °C. PCR products were separated on a 1,2 % agarose gel.
  • the vector pT7-TACS (SEQ ID NO: 16) was constructed for the expression of a TAT-Cre fusion protein in E. coli.
  • the plasmid contains the coding region of the 11 amino acid protein transduction domain of the wild-type HIV TAT protein (Green and Loewenstein, Cell, 55(6): 1179-88 (1988); Frankel and Pabo, Cell, 55(6): 1189-93 (1988); SEQ ID NO: 10) fused to the N-terminus of Cre recombinase protein sequence.
  • the 10-amino-acid strep tag at the C-terminus allows the detection and purification of the fusion protein using specific antibodies (Schmidt and Skerra, 3. Chromatogr A 676: 337-345 (1994)).
  • the protease factor Xa recognition site (Ile-Glu-Gly-Arg) permits the removal of the strep tag by proteolytic cleavage.
  • the estimated molecular weight of the TAT-Cre fusion protein is 42 kDa.
  • a scheme of the TAT-Cre expression vector is depicted in figure 2.
  • the E. coli strain BL21(DE3)-RIL (Stratagene) was used. This strain carries an IPTG-inducible T7 polymerase gene and additional copies of the tRNA genes for the Yare codons' argU, ileY and leuW.
  • E. coli BL21(DE3)-RIL cells were transformed with pT7-TACS and grown in LB medium containing 100 ⁇ g/ml ampicillin.
  • the expression of the 40 kDa TAT-Cre fusion protein could be strongly induced by the addition of 0,5 mM IPTG to the culture medium.
  • Analysis of protein lysates revealed that approximately 50 % of TAT-Cre protein accumulated as insoluble inclusion bodies. The inclusion bodies where extracted and dissolved in 8 M urea. TAT-Cre was subsequently purified from this fraction using ion exchange chromatography. The quantity and purity of TAT-Cre protein was determined using Coomassie stained SDS-PAGE gels and Western blot analysis (figure 3).
  • TAT-Cre protein extracts of 64 % purity and a concentration of 100 ⁇ g/ml.
  • fibroblast cell line M5Pax8 R. K ⁇ hn, unpublished
  • Cre recombinase allows the expression of a ⁇ -galacosidase gene (Buchholz et al, Nucleic Acids Res. 24, 4256-4262, 1996).
  • TAT-Cre protein To investigate the activity of TAT-Cre protein in a living organism, we used a transgenic mouse strain carrying a loxP-flanked target for Cre-mediated recombination (Thorey et al., 1998, Mol. Cell. Biol. 18: 3081 - 3088). Mice where treated three times with intraperitoneal injections of 75 ⁇ g TAT Cre protein at two-day-intervals and analysed 2 days later. Genomic DNA was isolated from a variety of organs and subjected to PCR amplification which specifically amplifies a 400 bp fragment of the recombined allele. The deleted allele could be detected in multiple tissues from treated mice indicating TAT-Cre-mediated recombination in these organs (figure 6). This experiments demonstrates that TAT-mediated delivery of active Cre protein works with sufficient efficacy to facilitate inducible gene targeting in cell lines and in living organisms.
  • the vectors pT7-VPCS (SEQ ID NO: 17) and pCRT7- ⁇ VPCS (SEQ ID NO: 15) were constructed for the expression of VP22-Cre and ⁇ VP22-Cre fusion proteins in E. coli.
  • the VP22-Cre gene of pT7-VPCS contains the full length protein translocation domain of the HSV VP22 protein (Elliott and O'Hare, Cell, 88(2): 223-33 (1987), whereas the ⁇ VP22-Cre gene of pCRT7- ⁇ VPCS contains a truncated VP22 protein transduction domain (amino acids 159 - 301; Invitrogen; aa 16-157 of SEQ ID NO:14) fused to the N-terminus of Cre recombinase protein sequence.
  • a 10-amino-acid strep tag at the C- terminus of Cre protein sequence allows the detection and purification of the fusion proteins using specific antibodies (Schmidt and Skerra, 3.
  • the protease factor Xa recognition site permits the removal of the Strep tag by proteolytic cleavage.
  • the estimated molecular weight is 75 kDa for VP22-Cre protein and 60 kDa for ⁇ VP22-Cre protein.
  • a scheme of the vectors pT7-VPCS and pCRT7- ⁇ VPCS is depicted in figure 7.
  • E. coli BL21(DE3)-RIP cells (Stratagene) were transformed with pT7-VPCS and cultured in LB medium containing 100 ⁇ g/ml ampicillin.
  • E. coli BL21(DE3)-pLysS cells (Stratagene) were transformed with pCRT7- ⁇ VPCS and cultured in LB medium containing 25 ⁇ g/ml kanamycine and 34 ⁇ g/ml chloramphenicol.
  • Expression of the VP22-Cre and ⁇ VP22-Cre fusion proteins could be induced by the addition of 0,5 mM IPTG to the culture medium.
  • the fibroblast cell line M5Pax8 that contains a loxP- containing reporter construct.
  • Cre recombinase the reporter allows the expression of a ⁇ -galacosidase gene (Buchholz et al, Nucleic Acids Res. 24, 4256-4262, 1996).
  • the cells where cultured for 18 h with increasing concentrations of VP22-Cre and ⁇ VP22-Cre in serum-free medium and analysed 4 days later for ⁇ -Galacosidase activity.

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Abstract

L'invention concerne l'utilisation d'une protéine de fusion comprenant un domaine d'ADN recombinase spécifique de site et un domaine de transduction de protéines pour la préparation d'un agent afin d'induire une modification du gène cible dans un organisme vivant ou dans des cellules cultivées. L'invention concerne également des protéines de fusion appropriées et une méthode de production desdites protéines de fusion.
PCT/EP2001/000060 2000-01-07 2001-01-05 Transduction de recombinases pour ciblage genetique inductible WO2001049832A2 (fr)

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CA002396149A CA2396149A1 (fr) 2000-01-07 2001-01-05 Transduction de recombinases pour ciblage genetique inductible
AU33683/01A AU3368301A (en) 2000-01-07 2001-01-05 Transduction of recombinases for inducible gene targeting
JP2001550361A JP2003518947A (ja) 2000-01-07 2001-01-05 誘導可能遺伝子標的化のためのレコンビナーゼのトランスダクション
IL15051001A IL150510A0 (en) 2000-01-07 2001-01-05 Transduction of recombinases for inducible gene targeting
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EP00100351A EP1118668A1 (fr) 2000-01-07 2000-01-07 Transduction d'une recombinase pour le ciblage inductible de gènes
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WO2003070931A2 (fr) * 2002-02-21 2003-08-28 Vision 7 Gmbh Techniques de recombinaison d'adn a des sites specifiques
EP1342781A1 (fr) * 2002-03-09 2003-09-10 ARTEMIS Pharmaceuticals GmbH Protéine de fusion de recombinase avec une assimilation cellulaire accrue
WO2003076561A2 (fr) * 2002-03-09 2003-09-18 Artemis Pharmaceuticals Gmbh Proteine de fusion a recombinase presentant un apport cellulaire ameliore
US6773920B1 (en) 1999-03-31 2004-08-10 Invitrogen Corporation Delivery of functional protein sequences by translocating polypeptides
CN100410369C (zh) * 2006-06-22 2008-08-13 复旦大学 经tat和nls多肽修饰的φc31整合酶蛋白及其应用
EP2120987A1 (fr) * 2007-03-02 2009-11-25 Arbor Vita Corporation Traitement des incidents cérébrovasculaires et d'autres maladies sans inhibition des canaux calcium de type n
WO2012160093A1 (fr) * 2011-05-23 2012-11-29 Novozymes A/S Intégrations simultanées spécifiques d'un site de multiples copies géniques dans un champignon filamenteux

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US8110719B2 (en) * 2008-04-25 2012-02-07 New York Blood Center, Inc. ABI1 conditional knockout mouse

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WO2000058488A2 (fr) * 1999-03-31 2000-10-05 Invitrogen Corporation Diffusion de sequences de proteines fonctionnelles par translocation de polypeptides
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6773920B1 (en) 1999-03-31 2004-08-10 Invitrogen Corporation Delivery of functional protein sequences by translocating polypeptides
WO2003070931A2 (fr) * 2002-02-21 2003-08-28 Vision 7 Gmbh Techniques de recombinaison d'adn a des sites specifiques
WO2003070931A3 (fr) * 2002-02-21 2003-11-27 Vision 7 Gmbh Techniques de recombinaison d'adn a des sites specifiques
EP1342781A1 (fr) * 2002-03-09 2003-09-10 ARTEMIS Pharmaceuticals GmbH Protéine de fusion de recombinase avec une assimilation cellulaire accrue
WO2003076561A2 (fr) * 2002-03-09 2003-09-18 Artemis Pharmaceuticals Gmbh Proteine de fusion a recombinase presentant un apport cellulaire ameliore
WO2003076561A3 (fr) * 2002-03-09 2004-01-22 Artemis Pharmaceuticals Gmbh Proteine de fusion a recombinase presentant un apport cellulaire ameliore
CN100410369C (zh) * 2006-06-22 2008-08-13 复旦大学 经tat和nls多肽修饰的φc31整合酶蛋白及其应用
EP2120987A1 (fr) * 2007-03-02 2009-11-25 Arbor Vita Corporation Traitement des incidents cérébrovasculaires et d'autres maladies sans inhibition des canaux calcium de type n
EP2120987A4 (fr) * 2007-03-02 2010-08-18 Arbor Vita Corp Traitement des incidents cérébrovasculaires et d'autres maladies sans inhibition des canaux calcium de type n
US8288345B2 (en) 2007-03-02 2012-10-16 Nono, Inc. Treating stroke and other diseases without inhibiting N-type calcium channels
US9061070B2 (en) 2007-03-02 2015-06-23 Nono, Inc. Treating stroke and other diseases without inhibiting N-type calcium channels
WO2012160093A1 (fr) * 2011-05-23 2012-11-29 Novozymes A/S Intégrations simultanées spécifiques d'un site de multiples copies géniques dans un champignon filamenteux
US9574199B2 (en) 2011-05-23 2017-02-21 Novozymes A/S Simultaneous site-specific integrations of multiple gene-copies in filamentous fungi
US9850501B2 (en) 2011-05-23 2017-12-26 Novozymes A/S Simultaneous site-specific integrations of multiple gene-copies

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JP2003518947A (ja) 2003-06-17
CA2396149A1 (fr) 2001-07-12

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