JP2005323619A - Delivery of functional protein sequences by translocating polypeptides - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for modulating a cellular process characterized in that a cell in culture is contacted with a cell process-modifying molecule attached to a translocating polypeptide. <P>SOLUTION: For example, in one embodiment, a cell in culture is transfected with a target gene by contacting the cell in culture with a polynucleotide (that contains the target gene) attached to a translocating polypeptide. In another embodiment, expression of a target gene product in a cell in culture that contains a target gene under control of one or more regulatory elements is modulated by contacting the cell in culture with one or more regulatory agents attached to a translocating polypeptide. The one or more regulatory agents are translocated into the cell in culture and interact therein with the one or more regulatory elements to modulate expression of the target gene product by the cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to methods for translocating polynucleotides and polypeptides between cells. More specifically, the present invention relates to the use of translocation proteins to deliver molecules that alter cellular processes into cells such that the molecules specifically interact with responsive target sites.

Background of the Invention Translocation proteins are defined by their ability to cross biological membranes such as cell membranes. Several translocation proteins have been described, for example VP22 derived from type I herpes simplex virus (G. Ellior and P. O'Hare, Cell 88, 223-233 (1997)), antennapedia derived from Drosophila Examples include fragments of proteins (Antp) (D. Derossi et al., Journal of Bioligical Chemistry 269, 10444-10450 (1994)) and protein H derived from Streptococcus pyrogenes (Axcrona et al., Manuscript in preparation (1999)).

  Antennapedia is a homeoprotein with a DNA binding domain that is composed of three alpha helices, with a beta turn separating helices 2 and 3. Experiments have shown that a 16 amino acid peptide corresponding to the third helix, called Antp, can translocate across the membrane and accumulate in the cytoplasm and nucleus (Derossi et al., Supra). The peptide is internalized at temperatures as low as 4 ° C., suggesting that endocytosis is not involved in the internalization of the peptide. Furthermore, since translocation does not require classical endocytosis, Antp does not migrate through endosomal and lysosomal compartments. Therefore, Antp is resistant to proteolysis and has enhanced activity in most cell compartments (D. Derossi et al., J Biol Chem 271: 18188-18193, 1996).

  Recent experiments showing that a helix composed of a reverse helix (ie, a reverse primary sequence) and a D-enantiomer can cross the cell membrane at 4 ° C. indicates that the internalization of Antp is the formation of reversed micelles in phospholipid bilayers. Is suggested to enter the cell without receptor dependence and energy (H. Hall et al., Current Biol 6: 580-587, 1996).

  The usefulness of Antp as a vector peptide is due to the genetic fusion of Antp to various peptides of interest (F. Perez et al., J Cell Sci 102: 717-722, 1992, F. Perez et al., Mol Endocrinol 8: 1278 -87, 1994, and A. Prochiantz, Curr Opinion Neurob 6: 629-634, 1996), or covalently via a cysteine residue (D. Derossi et al., Supra). Internalization of peptides as long as 41 amino acids and internalization of charged phosphopeptides (B. Allinquant et al., J Cell Biol 128: 919-927, 1995) has been demonstrated in neurons. In each case, the sequence fused to Antp retained the expected biological function. In addition, Antp is the only translocating peptide used to deliver oligonucleotides (up to 45 nucleotides in length) to cultured cells (CM Troy et al., J Neuro 16: 253-61, 1996, G. Elliot Et al., J Virol 172: 6448-6455, 1998).

  Protein H is a surface antigen of Streptococcus pyrogenes, a human pathogen. Protein H is taken up by B and T lymphocytes and translocated to the nucleus. In contrast to other translocated proteins that do not appear to affect cell function, protein H has a cytostatic effect believed to be the result of its association with nucleoprotein SET and hnRNP A2 / B1 (D. Derossi et al., Supra). To date, the translocation of protein H bound to other molecules has not been proven.

  The best studied translocation protein is the herpes simplex virus protein VP22, which has a unique ability to translocate between cultured mammalian cells. When cells are transfected with a plasmid encoding VP22 protein, the expressed protein accumulates in the cytoplasm of the transfected cells and diffuses to surrounding untransfected cells by translocation across the cell membrane And accumulate in the nucleus. This process occurs even at 4 ° C, does not require energy, and appears to be independent of endocytosis. If the protein that moves through the cell is blocked by Brefeldin A, transport of VP22 can still occur. Studies on the cytoskeletal elements during VP22 migration suggest that the actin cytoskeleton is involved in VP22 translocation (Elliot and O'Hare, supra).

  Delivery of several functional VP22 fusion proteins has been described, such as VP22-p53 (A. Phelan et al., Nature Biotechnology 16: 440-443, 1998) and VP22-thymidine kinase. (MS Dilber et al., Gene Therapy 6: 12-21, 1999). At least 20 different mammalian cell types can incorporate functional VP22-GFP fusion proteins (Elliot and O'Hare, supra, Aints A. et al., J. Gene Med. 1: 275-9, 1999 and Wybranietz WA et al., J. Gene Med. 1: 265-274, 1999), such cell types include, for example, mouse skeletal myoblasts that resist conventional transfection techniques (Derer W. et al. J. Mol. Med. 77: 609-6138, 1999).

  Transfection of cells with plasmid DNA has been an invaluable tool for biological system studies. Various transfection methods (eg, calcium phosphate method) exist on the market, but using these methods, more than 50% of the cells express the genes carried on the plasmid transfected into the cells That is rare. Since most cells take up exogenous DNA, inefficient transfection does not appear to be due to the inability of the DNA complex to enter the cell. Most DNA is internalized by endocytosis with very few internalized DNAs, reaching the cytoplasm or nucleus where expression occurs. Indeed, observation of directly injected lipid-DNA complexes suggests that the transfer from the endosome to the cytoplasm and nucleus is the most important constraint for successful transfection (JH Richardson et al., Proc Natl. Acad. Sci. 92: 3137-3141, 1995). Consistent with this observation, peptides having membrane fusion activity, such as hemagglutinin fusogenic peptides (J. Zabner et al., Journal of Biological Chemistry 270: 18997-9007, 1995), or nuclear targeting sequences (M. Wilke et al., Gene Therapy 3, 1133-1142 (1996)) may increase transfection efficiency.

Thus, in the art, a new and better method for regulating the expression of target genes in cells, as well as the major block for the expression of transfected genes (ie, degradation in endosomes and the inability of DNA to enter the cell nucleus). There is a need for transfection reagents and methods for their use to overcome the problem.
(G. Ellior and P.O'Hare, Cell 88, 223-233 (1997)) (D. Derossi et al., Journal of Bioligical Chemistry 269, 10444-10450 (1994)) Axcrona et al., Manuscript in preparation (1999)

Brief Description of the Invention The present invention translocates a cultured cell into a cultured cell by contacting the cultured cell under appropriate conditions with a cellular process-modifying molecule bound to a translocating polypeptide, where the molecule A method for modulating cellular processes in cells in culture is provided, characterized by modulating cellular processes in the cells by specifically interacting with target sites responsive to It overcomes these problems in the industry.

  In another embodiment, the present invention provides a method for transfecting a cultured cell with a target gene, the method comprising culturing the cultured cell with a target bound to a translocating polypeptide under appropriate conditions. Transfecting the cell with the target gene by contacting with a polynucleotide containing the gene.

  In yet another embodiment, the present invention provides a method for modulating the expression of a target gene product in cultured cells transfected with a target gene under the control of one or more regulatory elements, The method involves translocating the one or more modulators into a cell by contacting the cell with one or more modulators bound to a translocating polypeptide under appropriate conditions, wherein the one or more modulators are translocated into the cell. By regulating the expression of the target gene product by the cell by interacting with the regulatory elements of

  In yet another embodiment, the present invention provides a vector comprising a polynucleotide encoding a molecule that alters a cellular process linked to a translocating polypeptide.

Detailed Description of the Invention According to the present invention, under appropriate conditions, a cultured cell is contacted with a cellular process-modifying molecule bound to a translocating polypeptide, thereby translocating the molecule into the cell. There is provided a method for modulating a cellular process, characterized by modulating the cellular process in the cell by specifically interacting the molecule with a responsive target site.

  As used herein, the term “translocating protein” means a protein, polypeptide, or functional fragment thereof that crosses a biological membrane. Translocated proteins, polypeptides, functional fragments and homologues thereof have the following properties. That is, resistance to proteolysis, penetration into cell membranes independent of receptors, and penetration into cell membranes that do not substantially require energy. Representative translocation proteins that can be used in the methods and constructs of the present invention include VP22 derived from herpes simplex virus type 1 (G. Elliot and P. O'Hare, 1997, supra), and antennas derived from Drosophila. Fragment of pedia protein (Antp) (amino acids 43 to 58) (5'-RQIKIWFQNRRMKWKK-3 ') (SEQ ID NO: 21) (Axcrona et al., Supra, 1999), protein H derived from Streptococcus pyrogenes (D. Derossi et al. , J. Biol. Chem. 271: 18188-93, (1996)). Each translocation protein has different properties, but the general use of translocation proteins is by constructing a fusion molecule (e.g., a fusion protein) or by attaching the desired molecule to the translocation protein (e.g., covalently or via a linker). To deliver other molecules to the cell by binding. In the fusion protein, the translocated protein can be located at its N-terminus or C-terminus. A preferred fusion protein or polypeptide for use in practicing the methods of the invention is a VP22 polypeptide.

  As used herein, the term “VP22 polypeptide” refers to the herpesvirus VP22 protein, as well as functional fragments thereof having the translocation properties of the intact protein. Furthermore, as used herein, the term “VP22 polypeptide” refers to VP22 protein homology, including those derived from varicella-zoster virus (VZV), equine herpesvirus (EHV), bovine herpesvirus (BHV), etc. Bodies, fragments having their transport activity (ie “functional”), mutants and chimeric combinations.

  In particular, VP22 polypeptides include polypeptides corresponding to amino acids 60-301 and 159-301 of the full-length HSV1 VP22 sequence (1-301), whose sequence is disclosed in FIG. 4 of WO 97/05265. . Homologous proteins and fragments based on the sequences of VP22 protein homologues derived from other herpesviruses are described in US Pat. No. 6,017,735, which is hereby incorporated by reference in its entirety.

  As used herein, the term “fusion protein” refers to two different proteins, polypeptides, peptides, and / or fragments that are encoded by the same reading frame, usually not naturally associated with each other, together. Results in two or more different proteins and / or fragments that are “fused” together. The fusion protein used in the methods of the invention is produced from a nucleotide sequence that encodes a translocating polypeptide (eg, a VP22 polypeptide) and another functional peptide in the same reading frame. The polynucleotide encoding the fusion protein may also contain additional peptide or polypeptide sequences useful in the methods of the invention (e.g., sequences encoding epitope tags, sequences encoding affinity purification tags, additional functions) in the same reading frame. A sequence encoding a sex protein, etc., or any combination of two or more thereof).

  In one embodiment, the present invention provides a method for transfecting a cell with a target gene, the method comprising, under suitable conditions, the cell comprising a target gene bound to a translocating polypeptide. Transfecting the cell with the target gene by contacting with a nucleotide. As used herein, the term “transfected” means that a gene that has been translocated in a cultured cell due to the translocation properties of the associated translocating polypeptide is at least transiently expressed in the cell, ie, the It means that the cell is transiently transfected by the target gene.

  The size of a polynucleotide that can be transfected into a cell according to the methods of the present invention ranges from about 10 nucleotides to about 10 kilobases (kb). For example, polynucleotides ranging from about 20 nucleotides (nt) to about 5 kb, or from about 100 to 500 nt can be transfected into cells using the methods of the invention. Generally, the target polynucleotide is transiently transfected into a cell population in culture (eg, monolayer or tissue culture). Conventional means used to aid transfection or transduction, such as electroporation, infection with viral vectors, calcium phosphate transfection, dextran sulfate transfection, lipofection, cytofection, particle bead bombardment, etc. None are required. Instead, all that is required is a purified translocated protein or synthetically prepared having a polynucleotide of interest linked to the translocated protein by a covalent bond or linker molecule as described herein. Contact (ie, co-culture) of the translocated protein with the cell population to be transfected. Any type of prokaryotic or eukaryotic cultured cell can be transfected using the methods of the present invention, such cells include, for example, mammalian cells, yeast cells, insect cells or plant cells. However, it is presently preferred that the cultured cells are monolayers of mammalian cells or insect cells.

  In the methods of the invention where the translocated protein is bound to plasmid DNA (i.e., bound via covalent or non-covalent interactions), the DNA may be delivered to the nucleus for gene expression. it can. Delivery of DNA using the translocation proteins described herein is a very useful research tool. In up to 100% of the cells in which a desired polynucleotide containing an open reading frame (e.g., a polynucleotide contained in a plasmid) is delivered by the translocation protein of the present invention, the polynucleotide is internalized and transported to the nucleus. The open reading frame is expressed, thus obtaining a uniform population of cells for studying cellular processes such as cell cycle regulation, transcriptional regulation, translational regulation.

  In another embodiment according to the present invention, a method is provided for modulating the expression of a target gene product in a cultured cell containing the target gene under the control of one or more regulatory elements. In this embodiment, the method of the invention comprises culturing the one or more modulators under suitable conditions by contacting the cells with one or more modulators bound to a translocating polypeptide. By modulating the expression of the target gene product by the cell by translocating into the cell, where it interacts with one or more regulatory elements.

  For example, a polynucleotide linked to a translocating polypeptide such as VP22 can be translocated into the nucleus of the cell for expression of all or part of the polynucleotide. In one embodiment, the polynucleotide comprises an open reading frame encoding a protein of interest (eg, a target gene product or reporter gene product). Alternatively, the polynucleotide may be a vector (eg, a plasmid with a supercoil) that contains a cloned open reading frame encoding a target gene.

  The translocation protein and associated cellular process-modifying molecule direct the translocation protein to the cytoplasm of the cultured cell population for expression and to the cell nucleus when the translocation protein is bound (e.g., fused) to a nuclear export signal (NES). It was found to be obtained. Recently, signals for the transport of proteins from the nucleus have been described. Analysis of PKI (thermal stability inhibitor of cAPK, cyclic AMP-dependent protein kinase A) (Y. Wang et al., Gene Therapy 4, 432-441 (1997)) and analysis of HIV Rev protein (W. Wen et al., Cell 82, 463-473 (1995)) showed sufficient leucine-rich sequences to induce heterologous sequences outside the nucleus and in the cytoplasm. Furthermore, fusion of NES to a heterologous protein containing the canonical SV40 large T antigen NLS has been shown to result in the distribution of the protein between the cytoplasmic and nuclear compartments (Wang et al., Supra). Similarly, Rev proteins contain sequences for both nuclear and nuclear export and are found in both cytoplasmic and nuclear compartments of cells (Wen et al., Supra). Thus, NES integration is a potential method for regulating nuclear targeting of translocated proteins such as VP22. This is especially because PKI NES can partially negate the very strong signal of SV40 NLS. When bound to a nuclear export signal, the translocated polypeptide and any bound polynucleotide can be stably introduced into the cytoplasm and nucleus of the cultured cell, thereby achieving partitioning of the polynucleotide between the cell compartments. it can. In the cytoplasm, the expression of the gene contained in the polynucleotide can be regulated using the methods of the invention described herein.

  Suitable nuclear export signals for practicing the present invention are known in the art and include, for example, nuclear export signals derived from HIV Rev proteins or thermostable inhibitors of cAPK. In many cases, a target target gene can be stably integrated into the genome of a cultured cell by including a nuclear export signal in a construct containing a translocated protein.

  By various methods, cultured cells can be contacted with translocation proteins bound to cell modifying molecules according to the present invention. In one method, an expressed cell population transfected with a polynucleotide encoding a translocation protein fused to a cell modifying molecule (e.g., as a fusion protein) is combined with the expressed translocation protein bound to the cell modifying molecule. Mix with the target cell population to be taken up spontaneously and co-culture. The expressed protein accumulates in the cytoplasm of the transfected expression cell, translocates across the cell membrane, diffuses to surrounding untransfected cells, and accumulates in its nucleus. For example, the expression cell may be a prokaryotic cell line such as E. coli and the target cell line may be any eukaryotic, such as a mammalian cell line such as CHO or COS, or an insect cell line such as Drosophila S2. It may be a cell line.

  Alternatively, the expressed cell population is cultured under conditions that promote the expression of the transfected gene and transfected expression using methods known in the art and described in the examples herein. A cell lysate of the cell population can be prepared and the lysate added to the cultured target cell population. When the translocating polypeptide is VP22, VP22 or a fusion protein comprising VP22 translocates to substantially 100% of the nucleus of the cell population. Culturing the target cell with a molecule containing a purified translocation protein or a synthetically prepared molecule containing a translocation protein linked to a polypeptide or nucleotide by a covalent bond or linker as described herein. You can also The translocating polypeptide and bound molecule are translocated throughout the cultured cell population within about 10 minutes to about 72 hours, more typically within about 10 minutes to about 50 hours, preferably about 10 minutes to about 24 hours. To do. However, uptake of the translocating polypeptide and bound molecules by the entire cell population may not take as long as about 10 minutes.

  A fusion of a translocation polypeptide and a known DNA binding protein can be used to deliver DNA (eg, a plasmid) containing an open reading frame to tissue culture cells. In this embodiment of the method of the invention, the DNA binding protein serves as a linker to join the translocating protein to a cell modifying polynucleotide (ie, a plasmid containing a polynucleotide that serves to modify a cellular process). Examples of protein linkers that can be fused to translocation proteins for delivery of polynucleotides such as plasmid DNA include histone 1 (H1) protein (M. Wilke et al., Supra, and Niidome et al., J. Biol. Chem. 272 , 15307-15312 (1997)) and non-histone protein HMG-17 (high-speed movement group 17) (SV Zaitsev et al., Gene Ther. 4, 586-592 (1997)). The interaction between DNA and HMG-17 has been thoroughly studied, demonstrating that HMG-17 interacts with DNA in a non-cooperative, non-specific and reversible manner (M Bottger et al., Arch, Geschwulstforsch 60, 265-270 (1990)). In either case, the entire DNA binding protein or a functional fragment thereof (that is, a fragment having DNA binding activity) can be used.

  Prior to mixing with the translocation protein or translocation protein-DNA binding domain fusion, it may be preferable to complex the DNA with a reagent such as polyethyleneimine (PEI) that condenses and neutralizes the charged DNA. unknown.

  Alternatively, if a shorter peptide linker is advantageous in the particular system used, this peptide linker can be used as a chemically synthesized peptide or as a nucleotide encoding a fusion protein expressed in a prokaryotic expression system to the translocation protein. Can be merged. Examples of short peptide sequences that can be fused to translocation proteins as chemically synthesized peptides or as fusion proteins include sequences comprising a polylysine sequence and three or more repeats of the peptide sequence LARL, such as LARL-LARL- LARL (SEQ ID NO: 3) is mentioned (JD Fritz et al., Hum. Gene Ther, 7: 1395-1404 (1996)). In some cases, as described herein, 3 to about 100, typically 3 to about 50 repeats of a LARL sequence can be used as a linking peptide, and 3 to Up to about 20 repeat sequences are currently preferred.

  A preferred linker for attaching the translocation protein to the cell-modified polynucleotide is the vaccinia virus topoisomerase I protein, or a mutant thereof, which allows the formation of a stable topoisomerase I-DNA conjugate. Vaccinia DNA topoisomerase is a eukaryotic type I topoisomerase (I) encoded by a virus consisting of 314 amino acids, which binds to double-stranded DNA and cleaves the phosphodiester backbone of one strand (S. Shuman and B Moss, Proc. Natl. Acad. Sci. USA 84: 7478-7482 (1987)). This enzyme, like the restriction endonuclease, exhibits a high level of sequence specificity. Cleavage occurs at 5 ′-(C / T) CCTT-3 ′, a common pentapyrimidine element in the fragile strand (S. Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699 ( 1994), JM Clark, Nucleic Acids Res. 16: 9677-9686 (1988) and SG Morham and SJ Shuman, Biol. Chem. 267: 15984-15992 (1992)). In the cleavage reaction, the binding energy is conserved through the formation of a covalent adduct between the 3 ′ phosphate of the broken chain and the tyrosine residue of the protein (Tyr-274). Vaccinia topoisomerase can religate covalently held strands beyond the same bond that was originally cleaved (which occurs during DNA relaxation) or religate to heterologous acceptor DNA. , Thereby making a recombinant molecule. When coupled to the translocation protein of the present invention, the vaccinia topoisomerase I linker binds to a double stranded oligonucleotide with a single 5'A base overhang, as made in Taq-mediated PCR. Such topoisomerase I-DNA conjugate can then be introduced into the cell.

  FIG. 7 shows a suitable vector in which a vaccinia topoisomerase I linker is used to attach the translocating protein to the double stranded oligonucleotide of interest. The vector pVP22 / Myc-His TOPO® (SEQ ID NO: 2) is a vector that binds VP22 to a double-stranded PCR product (ie, a cellular process modified oligonucleotide) having a single 5′A base overhang. A vaccinia topoisomerase I linker is used to generate a VP22 fusion with DNA. Such topoisomerase I-DNA conjugate can then be introduced directly into the cell.

  In another embodiment, translocation proteins are used with cationic liposomes to increase the efficiency of plasmid delivery. Fusion of translocated proteins to protein domains that readily associate with cationic liposomes, such as hydrophobic transmembrane domains or glycosylphosphatidylinositol (GPI) anchors, facilitates interaction at the lipid-DNA interface. After endocytosis of the liposome-DNA complex, the translocated protein translocates the complex through the endosomal membrane into the cytoplasm and ultimately into the nucleus for gene expression. Translocation proteins can also be used with compounds such as chloroquine that inhibit lysosomal hydrolases to enhance transfection efficiency (Niidome et al., J. Biol. Chem., 272: 5307-12, 1998).

  Polynucleotides encoding fusion proteins can be synthesized using standard molecular biology techniques (J. Sambrook, EF Fritsch and T. Maniatis (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ), Transfected into tissue culture cells, and tested for transposition ability using suitable methods known in the art such as immunofluorescence. See also the methods described in the examples herein.

  An inducible system is used to study the phenotypic effects of protein expression. Inducible systems allow the expression of proteins on demand, so such systems can be used as research tools to study cellular processes and even to allow the expression of toxic proteins in tissue culture. Can be used. Current inducible mammalian expression systems are derived from a variety of organisms, such as E. coli (U. Fischer et al., Cell 82: 475-483 (1995)), or Drosophila (M Transcription elements from Gossen et al., TIBS 18: 471-475 (1993)) are used with vectors containing promoters that respond to transcriptional regulators. Upon addition of the effector molecule, transcriptional regulator binding to the inducible promoter occurs, thus switching on gene expression.

  The present invention provides a novel approach to this problem by providing a method for modulating the expression of a target gene product in mammalian cells transfected with a target gene under the control of one or more regulatory elements. provide. In the method of the present invention, under appropriate conditions, a target cell is contacted with one or more modulators bound to a translocation polypeptide, and the one or more modulators are translocated into a mammalian cell, Thus, expression of the target gene product by the cell is regulated by interacting with one or more regulatory elements. The translocation polypeptide used in the methods of the invention for modulating the expression of a target gene product can be any translocation polypeptide disclosed herein, but is preferably a VP22 polypeptide.

  The modulator may be a polynucleotide, protein or polypeptide, or small molecule. For example, the regulatory element may be a promoter operably linked to the target gene, in which case the translocation of the regulator transactivates the expression of the target gene product by the promoter. The regulatory substance is preferably specific to the promoter, such as a polymerase specific to the promoter.

  An exemplary inducible system according to the present invention utilizes the bacteriophage T7 RNA polymerase that has been used to direct gene expression in mammalian cells. Expression of T7 RNA polymerase (T7 RNAP) by vaccinia virus (A. Ramsey-Ewing and B. Moss, J. Biol. Chem. 271: 16962-16966, 1996, TR Fuerst et al., Proc. Natl. Acad. Sci. 83 : 8122-8126, 1986) or expression of T7 RNA polymerase in stable cell lines (O. Elroy-Stein and B. Moss, Proc. Natl. Acad. Sci. 87: 6743-6747, 1990 and A. Lieber et al., Nucleic Acids Res. 17: 8485-8493, 1989), or introduction of T7 RNAP protein at the time of transfection (X. Chen et al., Cancer Gene Ther. 2: 281-289, 1995 and X. Chen et al., Nucleic Acids Res. 22: 2114-2120, 1994) promotes the specific expression of genes located downstream of the small T7 promoter. The specificity that T7 RNAP has for the T7 promoter ensures that the desired gene is expressed and non-specific gene activation does not occur. Expression with T7 RNAP has been reported to be very strong and may be 6 times higher than the RSV promoter (A. Lieber et al., Nucleic Acids Res. 17: 8485-8493, 1989). Furthermore, gene expression can be directed by T7 polymerase in the cell nucleus (Lieber, supra and J.J. Dunn et al., Gene 68: 259-266, 1988) or cytoplasm. These features suggested that T7 RNAP can be used to specifically regulate gene expression by adding a T7 RNAP / VP22 fusion protein to cells containing the T7 promoter construct. Direct delivery of T7 RNAP using VP22 technology allows for specific control of gene expression and minimizes the negative effects of delivery to non-target sites. Thus, the method of the present invention enables studies on phenotypic effects of protein expression on demand. Due to the specificity of the inducible system of the present invention, the expression of toxic proteins can also be studied in tissue culture. As used herein, the term “toxic protein” refers to a protein that has a direct and intrinsic toxic ability to a biological system and includes transdominant variants in the protein that result in a constitutively active form of the protein. Thus, toxic proteins are distinguished from “prodrug” type molecules that require post-expression modification to release toxic potential. Non-limiting examples of toxic proteins used in the practice of the methods of the invention are various oncogene products such as Raf and Ras oncogene products (reviewed in Avruch et al., Trends in Biology 19: 279-83, 1994). Have been).

  Alternatively, the modulator may be a transcription factor specific for the regulatory element such that translocation of the regulator transactivates the expression of the target gene product. For example, translocation proteins can be fused to a DNA binding domain such as from a Gal4 protein and a common transactivation domain such as VP16 or B42. In this embodiment of the invention, the fusion protein comprising the translocation protein is localized to the nucleus and then specifically activates a promoter comprising an upstream binding site for the DNA binding domain incorporated into the fusion protein. To do.

  “DNA binding proteins” intended for use herein belong to the known class of proteins that can bind directly to DNA and facilitate the initiation or repression of transcription. Representative DNA binding proteins intended for use herein include transcriptional regulatory proteins (eg, transcription factors, etc .; see, for example, Conaway and Conaway, Transcription Mechanisms and Regulation, Raven Press Series on Molecular and Cellular Biology, Vol. 3, Raven Press, Ltd, New York, NY, 1994, T. Boulikas, Critical Reviews in Eukaryotic Gene Expression, 4 (2 & 3): 117-321, 1994, A. Klug, Gene 135: 83-92, 1993, WM Krajewska, Int. J. Biochem., 24: 1885-1898, 1992).

  Transcription factors intended for use herein as a source of such DNA binding domains include, for example, homeobox proteins, zinc finger proteins, hormone receptors, helix-turn-helix proteins, helix-loop- Examples include helix protein, basic Zip protein (bZip), and β ribbon factor. See, for example, S. Harrison, "A Structural Taxonomy of DNA-binding Domains," Nature, 353: 715-719. Suitable homeobox DNA binding proteins for use herein include, for example, HOX, STF-1 (Leonard et al., Mol. Endo., 7: 1275-1283, 1993), Mat α-2, INV, etc. Is mentioned. See also Scott et al., Biochem. Biophys. Acta, 989: 25-48, 1989. A 76 amino acid fragment containing the STF-1 homeodomain (corresponding to amino acids 140-215 described in Leonard et al., 1993) binds DNA as hard as wild type STF-1. Suitable zinc finger DNA binding proteins for use herein include Zif268, GLI, XFin, and the like. See also Klug and Rhodes, Trends Biochem. Sci., 12: 464, 1987, Jacobs and Michaels, New Biol, 2: 583, 1990 and Jacobs, EMBO J., 11: 4507-4517, 1992.

（配列番号４）
式中、Xは該DNA結合ドメイン内の非保存アミノ酸を示し、星印は、ほぼ普遍的に保存されているが、いくつかの同定されたホルモン受容体においては変異が認められたアミノ酸残基を示し、括弧内の残基は任意の残基である(従って、該DNA結合ドメインは最小で長さ66アミノ酸であるが、いくつかの追加の残基を含んでもよい)。そのようなDNA結合ドメインは、2-half部位認識部位に結合し、当業界では、該認識部位を含む応答エレメントの制御下で転写をトランス活性化することが知られている。 The DNA binding domain used in the methods of the invention can be obtained from a member of the steroid / thyroid hormone nuclear receptor superfamily or is substantially the same as that obtained from a member of the superfamily. Also good. Relevant are the DNA binding domains of virtually all members of the steroid / thyroid hormone nuclear receptor. Such domains consist of 66 to 68 amino acid residues and carry about 20 invariant amino acid residues (eg, 9 cysteines). The superfamily members are characterized as proteins containing these 20 invariant amino acid residues. The highly conserved amino acids of the DNA binding domains of the superfamily members are as follows:

(SEQ ID NO: 4)
Where X denotes a non-conserved amino acid within the DNA binding domain, and the asterisk is an amino acid residue that is almost universally conserved but mutated in some identified hormone receptors. The residues in parentheses are arbitrary residues (thus the DNA binding domain is a minimum of 66 amino acids in length, but may contain several additional residues). Such DNA binding domains bind to a 2-half site recognition site and are known in the art to transactivate transcription under the control of a response element containing the recognition site.

  The GAL4 DNA binding domain does not interact with the 2-half site DNA recognition site. The DNA binding domain of the yeast GAL4 protein contains at least the first 74 amino-terminal amino acids (see, eg, Keegan et al., Science 231: 699-704, 1986). Preferably, the first 90 or more amino terminal amino acids of the GAL4 protein are used, for example, the 147 amino terminal amino acid residues of yeast GAL4.

  Another DNA binding domain that can be used in the practice of the present invention is the Tet operon. Tetracycline induction systems are known in the art (e.g. Gossen et al., Proc. Natl. Acad. Sci. 89: 5547-5551 (1992), Gossen et al., TIBS 18: 471-475 (1993), Furth et al., Proc. Natl. Acad. Sci. 91: 9302-9306 (1994) and Shockett et al., Proc. Natl. Acad. Sci. 92: 6522-6526 (1995)).

  A transcriptional regulatory domain is one that activates transcription of a gene sequence operably linked to a response element that is responsive to the system of the invention (ie, a transcriptional activation domain) and a response element that is responsive to the system of the invention. There are two types of those that repress or deactivate transcription of functionally linked gene sequences (ie, transcription repression domains). In general, when the transcriptional regulatory domain attached to the translocated protein is a transcriptional activation domain, the ability of the system of the present invention to activate transcription of the target gene is enhanced. Transcription activation domains intended for use in the practice of the present invention can be obtained from a variety of sources and are known in the art.

  Such transcription activation domains are typically derived from transcription factors and comprise a contiguous sequence that functions to activate gene expression when combined with an appropriate DNA binding domain. For example, suitable activation domains are obtained from the N-terminal region of members of the steroid / thyroid hormone nuclear receptor superfamily, transcription factor activation domains (eg, VP16, GAL4, NF-κB or BP64 activation domains), etc. (E.g., M. Manteuffel-Cymborowska, Acta Biochim Pol. 46 (1) 77-89 (1999), T. Tagami et al., Biochem Biophys Res. Commun. 253 (2): 358-63 (1998), W. Westin, Adv Pharmacol, 47: 89-112 (2000)). The presently preferred activation domain for use in the practice of the present invention is derived from the C-terminal region of the VP16 protein.

  Examples of transcriptional repressor domains that can be used in the method of the present invention include those that suppress transactivation of gene expression. Representative transcriptional repressor domains suitable for use as transcriptional regulatory domains in the methods of the present invention include RAFT, CREM, MECP-2, SMRT, NcoR, mSin3A, RAR, TR, SMRTR, and the like.

  Another method in which the translocated protein can be used in an inducible expression system to regulate the expression of a target gene is by using a site-specific recombinant sequence that is a nucleic acid sequence that is specifically recognized by a particular site-specific recombinase. Gene fusions or in vitro covalent linkages. The term “site-specific recombinase” as used herein is an enzyme that catalyzes excision and / or recombination of nucleic acid sequences, and can form an intermediate complex with transfer sequence DNA in a recombination event. These enzymes recognize relatively short, unique nucleic acid sequences that serve as sites for both recognition and recombination. Particularly useful recombinases in the practice of this invention are those that function in a wide range of cell types. This is because such enzymes do not require any host-specific factors and do not require ATP to function.

  Two major families of site-specific recombinases from bacteria and unicellular yeast have been described. The integrase family and the resolvase / invertase family. In these recombinases, the site-specific recombinase-catalyzed strand exchange requires a covalent protein-DNA intermediate formed between the recombinase enzyme and the DNA strand, (1) cleavage and ( 2) happens in two steps of recombination. For these two recombinase families, the nature of the catalytic amino acid residue of the enzyme and the entry direction of the nucleophilic group are different. In the case of cleavage catalyzed by the invertase / resolvase family, the nucleophilic group hydroxyl is derived from serine and the leaving group is the 3′-OH of deoxyribose. For the integrase family, the catalytic residue is tyrosine and the leaving group is 5′-OH. In both recombinase families, the recombination step is the reverse reaction of the cleavage step.

Cre recombinase activity has been studied as a model system for integrase. Cre is a 38 kD protein isolated from bacteriophage P1. Cre catalyzes the recombination reaction at a 34 base pair stretch of nucleic acid called loxP. The loxP site has the sequence 5'-ATAACTTCGTATA GCATACAT TATACGAAGTTAT-3 '(SEQ ID NO: 5, underlined is the spacer region) and consists of two 13 base pair palindromic repeats adjacent to the 8 base pair core sequence. (Hoess et al., Proc. Natl. Acad. Sci USA 79: 3398, 1982 and US Pat. No. 4,959,217, the disclosures of these references are hereby incorporated by reference in their entirety). This repetitive sequence acts as a Cre binding site and the intersection is in the inner spacer core. Each repetitive sequence binds to one protein molecule and the DNA substrate (one strand) is cleaved to form a protein-DNA intermediate with a 3'-phosphotyrosine bond between Cre and the cleaved DNA strand. It seems. Crystallography and other studies have shown that four proteins and two loxP sites (each on a different DNA molecule) form a synaptic structure (the DNA is similar to a model of four holiday-linked intermediates) Subsequent exchange of a second set of strands suggests splitting the intermediate into recombination products (see Guo et al., Naure 389: 40-46, 1997). The asymmetry of the core region of the loxP recombination sequence is responsible for the direction of the recombination reaction. If two loxP sites on the same DNA molecule are in a directly repeated orientation, Cre will cut out the DNA between those two sites, leaving a single loxP site on the DNA molecule (Abremski Et al., Cell 32: 1301, 1983). This repetitive sequence thus acts as a Cre-specific binding site where the recombination intersection is present in the core.

  Because the loxP site is complex in size, it exists only in the genome of the P1 phage. Thus, by using the loxP site in the method of the invention, the enzyme will not cleave the transfer sequence within the sequence unless the transfer sequence is derived from the genome of the P1 phage. Cre activity in various cell backgrounds, such as yeast, is determined by Cre (Plant et al., Plant J. 7: 649-659, 1995, Dale and Ow, Gene 91: 79-85, 1990, Odell et al., Mol. Gen. Genet. 223: 369-378, 1990), or mammalian cells such as rodent and human cells (van Deursen et al., Proc. Natl. Acad. Sci. USA 92: 7376-7380, 1995, Agah Et al., J. Clin. Invest. 100: 169-179, 1997, Sauer and Henderson, New Biologist 2: 441-449, 1990) show that host-specific factors for activity are not required. (Sauer, Mol. Cell. Biol. 7: 2087-2096, 1987).

Cre protein also recognizes several mutant or mutant lox sites (mutated relative to the loxP sequence) such as loxB, loxL and loxR sites found in the chromosome of E. coli. Other mutant lox sites include loxP511 (5'-ATAACTTCGTATA GTATACATT ATACGAAGTTAT-3 '(SEQ ID NO: 6, underlined spacer region), loxC2 (5'-ACAACTTCGTATA ATGTATGC TATACGAAGTTAT-3' (SEQ ID NO: 7, underlined spacer) Region, U.S. Pat. No. 4,959,217. One skilled in the art can prepare additional variants of the loxP site, generally in two repetitive sequences containing site-specific recombination sequences. It only has a total of 1 to 3 point mutations.Cre catalyzes the cleavage of the lox site in the spacer region, creating a 6 base pair sticky end.Two 13 bp inverted repeat domains in the lox site Represents the binding site of the Cre protein, which may be different as long as Cre can recognize both lox sites, but the overhanging ends of the cleaved DNA cannot re-anneal to each other The two lox sites are their spacers If different in the region, Cre cannot efficiently catalyze the recombination event using these two different lox sites, which will depend on the extent and location of the mutation at the binding site. For example, the loxC2 site can be efficiently recombined at the loxP site because the two lox sites differ by a single nucleotide in the left binding site, so Cre is the present invention. In the case of the site-specific recombinase used in carrying out the method, the site-specific recombination sequence is a loxP site or a variant thereof recognized by the Cre enzyme.

An integrase family recombinase with a similar function is Flp, a recombinase identified in a strain of Saccharomyces cerevisiae containing 2μ circular DNA. Flp is an inverted repeat of two 13 base pairs adjacent to an 8 base pair core sequence called FRT (Flp recombination target site) (5'-GAAGTTCCTATTC TCTAGAAA GTATAGGAACTTC-3 '(SEQ ID NO: 8), underlined spacer) Recognizes a DNA sequence consisting of sequences. The third repetitive sequence follows the 3 ′ end of the native sequence but does not appear to be necessary for recombinase activity. The Flp gene has been cloned, expressed in E. coli and mammalian cells (PCT international patent application PCT / US92 / 01899, publication number WO 92/15694, the disclosure of which is hereby incorporated by reference) and purified. (Meyer-Lean et al., Nucleic Acids Res. 15: 6469, 1987, Babineau et al., J. Biol. Chem. 260: 12313, 1985, Gronostajski and Sadowski, J. Biol. Chem. 260: 12328, 1985).

  Like Cre, Flp is a bacterium (Huang et al., J. Bacteriology 179: 6076-6083, 1997), insect (Golic and Lindquist, Cell 59: 499-509, 1989, Golic and Golic, Genetics 144: 1693-1711, 1996), plants (Lyznik et al., Nucleic Acids Res 21: 969-975, 1993), and mammals (US Pat.Nos. 5,677,177 and 5,654,182), which are functional in Flp Show no need for host-specific factors for functionality.

  Other integrases that can be used in carrying out the methods of the invention are retroviral integrases, such as HIV and ASV integrase (reviewed in Annu. Rev. Microbiol. 53: 245-81, 1990). Is).

  When carrying out the method of the invention to regulate the expression of the target gene product, the site-specific DNA recombinase or integrase fused to the translocation protein is replaced with a recombinase recombination site specific for the recombinase or integrase used. Introduced by the methods described herein into cells transfected with a plasmid containing a transcriptional blocking sequence (eg, a transcription termination sequence) placed adjacent to and between the promoter and the open reading frame encoding the target gene To do. For example, when the recombinase is Flp, the recombinase site is a frt site, and when the recombinase is Cre, the recombinase site is a lox site. When the transfected cells are brought into contact with recombinase or integrase translocation protein, the transcription terminator is removed by the activity of the recombinase, and the gene of interest is expressed as shown in FIG.

  Thus, in the method of modulating a cellular process of the present invention, one or more regulatory elements may include a transcription blocking sequence adjacent to the recombinase recombination site, and the modulator is specific for the recombination site. It may be a recombinase, where the recombinase translocation regulates the expression of the target gene product by causing recombination at the recombination site.

  Alternatively, instead of including a pair of recombinase sites adjacent to the polynucleotide segment to be excised, the target cell and the genome of the target cell containing the plasmid containing the second recombinase site that pair with the genomic recombinase site are Of recombinase sites may be incorporated (or may be naturally occurring). When such a cell is contacted with a recombinase (eg, integrase) specific for the recombinase site (s) in the target cell, the recombinase translocation triggers a recombination event, and the target gene becomes a genomic recombinase. It is stably incorporated into the genome of the target cell at the site.

  A recombinase or integrase is introduced by mixing two cell populations, one expressing a translocation protein-enzyme (eg Flp or Cre) fusion and the other containing a heterologous gene. Alternatively, translocated protein-enzyme fusions may be produced in prokaryotic or eukaryotic expression systems, purified using known methods and methods described herein, and added to cells containing the heterologous gene. . The cell may either be transiently transfected with a heterologous gene or have a heterologous gene stably integrated into its genome.

  Alternatively, the regulator used in the method for regulating gene expression of the present invention may be an HIV Rev protein paired with a Rev regulatory element (RRE) as a regulatory element. As illustrated in Example 5 herein, increasing the amount of Rev protein delivered to target cells containing RRE increases the expression of functionally linked target genes.

  In another embodiment of the method of altering cellular processes of the invention, the protein molecule fused to the translocation protein is an Fv antibody fragment or a single chain antibody (sFv). Preferably, a polynucleotide encoding a fusion protein containing a translocation protein and sFv is introduced into cultured cells by the method described herein, and translocation to the cell nucleus and intracellular expression are performed. When sFv is specific for an antigen target associated with an intracellular mechanism involved in cell function, such as a target located in the cell nucleus, binding of sFv to the intracellular target causes cell function, e.g., Ras signaling ( O. Elroy-Stein, TR Fuerst, B. Moss, Proc. Natl. Acad. Sci. 86, 6126-6130 (1989)), membrane transport (O. Cachet et al., Cancer Research 58, 1170-1176 (1998)) Alternatively, viral replication (JH Richardson, JG Sodroski, TA Waldmann, WA Marasco, Proc. Natl. Acad. Sci. 92, 3137-3141 (1995)) may be disrupted. Other examples of intracellular targets that can be used in methods of constructing single chain antibodies to alter the cellular processes of the present invention include human kinases, transcription factors, proteins that control apoptosis, cell cycle regulators, Tumor proteins and the like are included.

  Accordingly, the one or more modulators used in the method of modulating a cellular process of the present invention may comprise Fv or sFv specific for a component of one or more regulatory elements in cultured cells (the regulatory element is native). In which Fv or sFv is translocated into the cell by the translocating protein and the antibody is bound to the above components, thereby expressing the expression of the target gene product. Adjust.

  For example, the intracellular process was modified by making a fusion protein containing the anti-ATF-2 sFv fused to VP22 and the transcriptional activation domain of VP16 (FIGS. 2A-D). ATF-2 belongs to the bZIP family of transcription factors, as a homodimer or as a heterodimer with, for example, Jun (S. Huguier et al., Molecular and Cellular Biology 18: 7020-7029, 1998) via the 8-bp ATF / CREB motif. To control gene expression. When the fusion protein is expressed in a reporter cell line with a reporter gene, eg, ATF-2 bound upstream of a bacterial luciferase gene, binding of the sFv in the fusion protein to the ATF-2 antigen in the cell nucleus (FIG. 2A -D) induces the expression of the ATF-2 sFv-VP16 fusion (FIG. 2B) but not the CREB sFv-VP16 fusion (FIG. 2C), resulting in the expression of the reporter gene. This experiment demonstrates that ATF-2 sFv is delivered to the cell nucleus where it binds to the ATF-2 antigen.

  As used herein, “Fv” refers to a genetically engineered fragment comprising a light chain variable region and a heavy chain variable region that are expressed as two chains but are chemically linked. , "SFv" means a genetically engineered molecule comprising a light chain variable region and a heavy chain variable region that are linked together by a suitable polypeptide linker into a genetically fused single chain molecule .

  The binding of the variable regions of the L and H chains in Fv may be non-covalent as described in Inbar et al., Proc. Natl. Acad. Sci. USA 69: 2659-62, 1972. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde.

  An example of a linker used to join two segments of Fv or to join two other proteins (eg translocation protein and DNA binding protein) is N-succinimidyl (4-iodoacetyl)- Aminobenzoate, sulfosuccinimidyl (4-iodoacetyl) -aminobenzoate, 4-succinimidyloxycarbonyl-α- (2-pyridyldithio) toluene, sulfosuccinimidyl-6- [α- Methyl-α- (pyridyldithiol) -toluamide] hexanoate, N-succinimidyl-3-(-2-pyridyldithio) -propionate, succinimidyl 6 [3 (-(-2-pyridyldithio) -propionamide] hexano Art, sulfosuccinimidyl 6 [3 (-(-2-pyridyldithio) -propionamide] hexanoate, 3- (2-pyridyldithio) -propionyl hydrazide, Elman reagent Bifunctional cleavable crosslinkers such as dichlorotriazine acid, S- (2-thiopyridyl) -L-cysteine, etc. Still other bifunctional linking compounds are U.S. Patent Nos. 5,349,066, 5,618,528, Nos. 4,569,789, 4,952,394 and 5,137,877, each of which is hereby incorporated by reference in its entirety.

  These linkers can be coupled to the purified protein using a number of protocols known to those skilled in the art, such as the methods described in Examples 1 and 2 (Pierce Chemicals “Solutions, Cross-linking of Proteins: See Basic Concepts and Strategies, “Seminar # 12, Rockford, IL”.

Preferably, the antibody used in the method of the present invention is sFv consisting of V _H and V _L chains linked by a peptide linker. These single-chain antigen binding proteins (sFv) are produced by constructing a structural gene consisting of DNA sequences encoding _VH and _VL domains linked by oligonucleotides. After the structural gene is inserted into an expression vector, the expression vector is introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single chain polypeptide with a linker polypeptide that bridges the two V domains. Methods for producing sFv are described, for example, in Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242: 423-426, 1988; Pack et al., Bio / Technology 11: 1271-77, 1993; Ladner et al., US Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety. These known methods can be modified to create a fusion protein consisting of sFv and a translocation protein, as described herein.

For example, the linker in sFv is a peptide having about 2 to about 60 amino acid residues, typically about 5 to about 40, preferably about 10 to about 30 amino acid residues. It's okay. This alternative is particularly advantageous when the ligand moiety is proteinaceous. For example, the linker moiety may be a flexible spacer amino acid sequence as known in single chain antibody studies. Examples of such known linker moieties include GGGGS (SEQ ID NO: 9), (GGGGS) _n (SEQ ID NO: 10), GKSSGSGSESKS (SEQ ID NO: 11), GTSGSSGKSSEGKG (SEQ ID NO: 12), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 13), GSTSGSGKSSEGKG (SEQ ID NO: 14), GTSGSSGKPGSGEGSTKG (SEQ ID NO: 15), EGKSSGSGSESKEF (SEQ ID NO: 16), SRSSG (SEQ ID NO: 17), SGSSC (SEQ ID NO: 18) and the like are included. A diphtheria toxin trypsin sensitive linker having the sequence MGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO: 19) is also useful. Alternatively, the peptide linker moiety may be VM or AM, or a structure represented by the general formula AM (G ₂ to ₄ S) xAM, where x is an integer from 1 to 11 (SEQ ID NO: 20) You may have. Other linker moieties are described, for example, by Huston et al., PNAS 85: 5879-5883, 1988; Whitlow, M. et al., Protein Engineering 6: 989-995, 1993; Newton et al., Biochemistry 35: 545-553, 1996; AJ Cumber Bioconj. Chem. 3: 397-401, 1992; Ladurner et al., J. Mol. Biol. 273: 330-337, 1997; and U.S. Pat. Are incorporated herein by reference.

  When the target gene in the cultured cell is a reporter gene, for example, as known to those skilled in the art, a non-coding gene encoding a detectable marker such as E. coli β-galactosidase gene, luciferase, or CAT. A case of an endogenous gene is also included in the scope of the present invention.

  In order to assist in the purification of the fusion molecule or the detection of expression induced by the use of the method of the invention, the polynucleotide encoding the reporter gene includes an antibody epitope (eg, derived from Myc), a fluorescent peptide, Or it is often convenient to include additional nucleotide sequences encoding protein tags, such as poly-His tags.

  Various methods can be used to attach the peptide or oligonucleotide molecule to the translocating protein. For example, translocation proteins can bind to macromolecules and proteins such as DNA, and combine to form two low molecular weight chemical affinities that form linkers useful in the production of fusion proteins or genes used in the methods of the invention. The sex ligand can be used to covalently conjugate to a polynucleotide encoding a translocating protein or translocating polypeptide for use in the methods of the invention. Two such low molecular weight chemical affinity ligands are salicylhydroxamic acid (SHA) and phenylboronic acid (PBA), which react rapidly to form reversible pH-sensitive covalent bonds (Figure 3A- C), providing a convenient linker for attaching the translocated protein to another protein or polynucleotide. For example, PBA-NTP (available from ProLinx, Seattle, WA) can be used to synthesize nucleic acid molecules containing PBA. Alternatively, in the case of a double strand, a nucleic acid molecule containing PBA can be labeled with PBA-ATP using an enzyme terminal transferase. SHA-NHS esters can also be used to link SHA to lysine residues present in translocated proteins. In this embodiment, the PBA junction molecule and the SHA junction translocation protein are covalently linked and added to the cell. Alternatively, other linkers, such as disulfide bonds (broken upon delivery to the cytoplasm) or bifunctional linker molecules (eg, as disclosed herein) can also be used. Covalent bonds using these linkers, or other linkers known to those skilled in the art and disclosed herein, provide a relatively stable bond between the translocating protein and other molecules.

In addition, several strong non-covalent molecular interactions can be used to generate translocated protein-containing complexes. For example, streptavidin binds to biotin very strongly (dissociation constant is approximately ^10-15 ). This strong affinity is usually used to bind the protein to the substrate, but can be used to form a linker that connects the cellular process modifying molecule to the translocating polypeptide. For example, a fusion protein comprising a VP22 translocation protein and streptavidin is made and complexed with a biotinylated oligonucleotide to form a linker that binds the cellular process-modified polynucleotide to the translocation polypeptide. Streptavidin binds to biotin as a tetramer (tetramer has a molecular weight of 60,000 daltons) and VP22 appears to act as a multimer, making this combination appropriate.

  Another polypeptide molecule that can be used as a linker to attach a cellular process modifying molecule to a translocating polypeptide for use in the methods of the present invention is a single stranded DNA binding protein (SSB) from E. coli. ). Only 21 amino acid residues of SSB (amino acid residues 2 to 22) appear to be involved in binding to ssDNA (ie, “functional fragment of SSB”). Furthermore, the binding of SSB to ssDNA is not sequence specific. Thus, a fusion protein comprising a translocation protein fused to a functional fragment of SSB is a very attractive linker for joining the translocation protein to oligonucleotides or plasmid DNA. Unlike the linker molecules above (ie those containing PBA and SHA, or streptavidin and biotin) that need to modify the oligonucleotide to bind to the translocation protein, the fusion protein containing SSB and the translocation protein is modified Since it can bind to no DNA, it offers the advantage of saving time and money.

The invention will now be described in more detail with reference to the following non-limiting examples.
Example 1
Introduction of VP22 fusion protein into cultured cells by transfection
The complete open reading frame encoding VP22 protein was cloned into the eukaryotic expression vector pcDNA3.1 / myc-His (Invitrogen, San Diego, Calif.) And the vector pVP22 / Myc-His (FIG. 5, SEQ ID NO: 1). )made. The fusion partner ORF can be inserted into this vector at the multiple cloning site located between the VP22 ORF and the sequence encoding the C-terminal anti-myc epitope and poly-His tag. Anti-myc epitopes facilitate detection of recombinant proteins using anti-myc antibodies, and poly-His tags are useful for purification. In addition, the vector used was modified by covalent coupling of vaccinia virus topoisomerase I protein to linearized vector DNA (eg, pVP22 TOPO® TA Cloning® Kit (Invitrogen)). . In this type of vector, the ORF of the gene product of interest (ie, “fusion partner”) was cloned into the vector as a PCR product. An example of a topoisomerase conjugation vector encoding such a VP22 polypeptide is the pVP22 / Myc-HisTOPO® vector (FIG. 7, SEQ ID NO: 2). In either case, the cultured cells were then transfected with a plasmid containing the VP22 gene fusion.

  In a typical transfection, COS or CHO cells were seeded in 6-well plates, grown to approximately 50% confluency and then transfected. For each well, 5 μg of DNA is diluted with 1.5 ml of OptiMEM medium (Gibco BRL, Chagrin Falls, OH), 15 μl of Pfx-6 lipid (Invitrogen) for COS cells, and for CHO cells. Mixed with 15 μl of Pfx-7 lipid (Invitrogen). The diluted DNA plus lipid was incubated with the cells at 37 ° C for 4 hours, then replaced with an appropriate medium, and further incubated at 37 ° C for 40 to 48 hours.

  Diffusion of the VP22 fusion protein from the transfected cells to the surrounding untransfected cells was detected by immunofluorescence using an antibody against the myc epitope tag. In a typical immunofluorescence experiment, transfected cells in one 35 mm well of a 6-well tissue culture plate are washed with phosphate buffered saline (PBS) and fixed by incubating in 2 ml of methanol for 5 minutes. did. Cells are washed 5 times with PBS (2 ml per wash), blocked for 15 minutes with PBS containing 10% fetal bovine serum (FBS), then 1: 500 with 1 ml PBS containing 10% FBS. An antibody against diluted myc epitope tag (Invitrogen) was added and incubated for 20 minutes. To bind the fluorescent molecule to the antibody, the cells were washed twice with PBS and diluted 1: 500 with 1 ml of PBS containing 10% FBS, goat anti-mouse Oregon Green conjugate (Molecular Probes, Eugene, OR; cat # O-6383) was added and incubated for 20 minutes. After further washing twice, the antigen-antibody complex was observed using an Olympus IX-70 fluorescence microscope equipped with a fluorescein isothiocyanate (FITC) filter.

Translocations of several VP22 fusion proteins thus prepared, such as those incorporating Aequorea victoria green fluorescent protein ( ho GFP), lacZ, or site-specific recombinase Flp as fusion partners, were achieved by this method. .
Example 2
Transfection of cells with gene fusion and subsequent mixing with untransfected cells
To demonstrate how VP22 can be used to modulate the expression of functional gene products, a system has been developed that delivers site-specific DNA recombinase Flp. COS cells expressing VP22-Flp recombinase fusion protein were prepared as described above and mixed with CHO cells transfected with the reporter plasmid pFIN4 / lacZ (FIG. 1). In the reporter plasmid, the DNA segment containing the transcription terminator, bovine growth hormone polyadenylation signal (Goodwin and Rottman, J. Biol. Chem. 267: 16330-4, 1992) is a frt site (recombination site recognized by recombinase Flp). ), And separates the CMV promoter and other operably linked reporter genes encoding β-galactosidase as a reporter. Cells transfected with pFIN4 / lacZ did not express β-galactosidase due to the presence of a transcription terminator located between the frt sites.

  To demonstrate that the expression of the reporter gene is controlled by translocation of the VP22-Flp recombinase fusion protein from one cell population to another, two populations of CHO cells were prepared, one of which is VP22- Transfected with a plasmid expressing the Flp recombinase fusion protein and the other with a plasmid expressing the VP22-GFP fusion protein. Transfection was performed as described above. 24 hours after the completion of transfection, cells were collected by trypsinization. These two cell lines were then mixed and incubated for an additional 24 hours before staining for β-galactosidase activity.

  CHO cells transfected with pFIN4 / lacZ expressed only β-galactosidase when mixed with COS cells expressing VP22-Flp fusion. In the presence of Flp recombinase, the DNA segment containing the transcription terminator was removed by recombination of the frt site and β-galactosidase was expressed. Incubation of a population of CHO cells transfected with a plasmid expressing the VP22-GFP fusion protein but not the VP22-Flp fusion protein resulted in no expression of β-galactosidase.

This experiment shows that the VP22-Flp fusion protein translocates between different mammalian cell types and that the functional Flp recombinase can be delivered to the cell as a fusion partner in the VP22 fusion protein.
Example 3
Transfection with cell gene fusion and subsequent preparation of cell-free lysates from transfected cells In these studies, cell-free lysates were obtained from cells transfected with pVP22 / myc-His as follows: It was prepared as follows. COS cells were grown to 50% confluency in a 100 mm dish (approximately 10 ⁶ cells). Cells were transfected with 20 μg pVP22 / myc-His DNA using Pfx-6. Forty hours after transfection, cell monolayers were washed twice with PBS, then scooped and collected in 10 ml PBS. Cells were centrifuged at 500 g for 5 minutes, PBS was aspirated from the cell pellet, and the cell pellet was frozen on dry ice. The frozen cell pellet was stored at −80 ° C. until the lysate was prepared. Add 0.5 ml of ice-cold lysis buffer (10 mM HEPES, pH 7.9, 400 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 5% glycerol) to the cell pellet. And thawed on ice. The lysate was then briefly vortexed and centrifuged at 10000 xg for 5 minutes at 4 ° C.

The entire supernatant was immediately added to 2 × 10 ⁵ cells in a 35 mm plate without removing the tissue culture medium. After incubating at 37 ° C. for 10 minutes, the medium was removed, and the VP22 / myc-His protein located in the cell nucleus was detected using the immunofluorescence method described above.

  Another method for detecting uptake of VP22 fusion protein into mammalian cells from cell-free lysates prepared from cells expressing the fusion protein utilizes Western blotting. In a typical Western blot experiment, HeLa, COS or CHO cells were plated on a 60 mm dish at 50% confluence. After applying the lysate, the cells were washed once with PBS and then with PBS containing 500 mM NaCl to remove proteins that non-specifically bound to the outside of the cells. The cells were treated with trypsin for about 5 minutes to dissociate from the plate and all remaining extracellular peptide was digested. The cells were solubilized and proteins were separated on a 4-20% glycine gel (Invitrogen, Carlsbad, CA). The separated protein was then transferred to nitrocellulose and probed with an appropriate antibody conjugated to horseradish peroxidase (HRP). Next, VP22 fusion protein was detected using chemiluminescence.

Thus, the VP22 / myc-His protein contained in the lysate of cells transfected with pVP22 / myc-His was translocated to the nucleus of all untransfected cells within 10 minutes of contact. This finding indicates that lysates containing VP22 are useful for delivering protein sequences to cell types without the need for transfection of receptor cell populations.
Example 4
Expression of VP22 fusion protein in E. coli and subsequent application of purified protein to cultured cells To allow expression and purification of VP22 fusion protein from E. coli, vector pCRT7 / VP22 -1 was developed. This vector utilizes a C-terminal fragment of VP22 protein (amino acids 159-301) that has been demonstrated to be sufficient for translocation of the VP22 fusion protein across the cell membrane. Using the method described above, VP22 fusion proteins containing various proteins (including HIV Rev protein and human protein rhoA) as fusion partners were prepared, and the fusion protein was expressed and purified. Following uptake by cultured cells, the activity of each fusion partner was demonstrated. Jurkat, known to be refractory to standard transfection protocols, to prove high efficiency using conventional techniques to allow transposition to occur in cell culture even when only a few cells are transfected Uptake of VP22 / GFP fusion protein by T-cells and PC12 cells was also performed. These experiments show that the VP22 fusion protein can be purified and then delivered to virtually all cells of the cultured mammalian cell population, making the cell line refractory to standard transfection protocols. Even if known to be, it completely eliminates the need for transfection.

  pCRT7 / VP22-1 is derived from the pET9b vector backbone (Novagen, Madison, Wis.). Sequences encoding the C-terminal region of VP22 (amino acids 159-301) sufficient for translocation activity in preparation of pCRT7 / VP22-1, fragments containing multiple cloning sites and myc and His tags from vector pVP22 / myc-His Was inserted into the pET9b vector backbone. The multicloning site of pCRT7 / VP22-1 was derived from that of pVP22 / myc-His. A pCRT7 / VP22-1 vector was prepared and coupled with vaccinia topoisomerase I in exactly the same manner as the preparation of the above-described pVP22 / myc-His-TOPO® plasmid. Thus, in this vector, the sequence encoding the fusion partner ORF can be inserted into one of the multiple cloning sites or used in the pVP22 / myc-His or pVP22 / myc-His-TOPO® plasmid. Can be cloned into the topoisomerase cloning site as a PCR product in a similar manner.

  In a typical experiment, the VP22 fusion protein was expressed as follows. 10 ng of pCRT7 / VP22-1 DNA was transformed into 50 μl of BL21 (DE3) plysS cells. Transformed cells were incubated in 200 μl SOC medium for 1 hour at 37 ° C., then diluted to 2 ml with Luria-Burtoni (LB) medium supplemented with 50 μg / ml kanamycin and grown overnight at 37 ° C. . A 2 ml culture was used to inoculate 50 ml of LB medium containing 50 μg / ml kanamycin. The cells were grown to an optical density of 0.5 to 0.6, and then continued to grow for 30 minutes either at 37 ° C. or lowered to room temperature (approximately 25 ° C.). A 1 ml culture was removed and continued to grow. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the remaining culture to a final concentration of 1 mM. Cells were grown for an additional 4 hours before gel samples were prepared from induced (plus IPTG) and non-induced cultures. 200 μl of each culture was removed, the cells were collected by centrifugation, and the pellet was placed in 50 μl of 1 × SDS / PAGE sample buffer. Alternatively, cells were harvested from the remaining culture by centrifugation and the cell pellet was stored at -80 ° C.

  The VP22 fusion protein was purified as follows. The cell pellet was thawed at ice temperature and resuspended in 4 ml ice-cold lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 5 mM imidazole). The following were added to the lysis buffer immediately before cell lysis: β-mercaptoethanol to 5 mM, α-toluenesulfonyl fluoride (PMSF) to 0.5 mM, leupeptin to 1 μg / ml each. And pepstatin, and lysozyme to 1 mg / ml. Lysates were incubated on ice for 20-30 minutes and then sonicated for 3 × 10 seconds while kept on ice. DNase and RNase were added to a final concentration of 10 μg / ml, respectively. The lysate was left on ice for an additional 20 minutes, then aspirated 3 times through a 21 gauge needle and centrifuged at 20000 g for 15 minutes. After centrifugation, a gel sample was prepared from the soluble supernatant. The supernatant is placed in a column containing 1 ml of Probond resin equilibrated with lysis buffer (Probond beads interact with 6-histidine array tagged proteins). The resin and supernatant were mixed in a column on ice for 1-2 hours. The column was then fixed vertically to precipitate the resin.

  A sample of the supernatant was removed and tested for the presence of unbound protein by SDS / PAGE. The resin was washed by passing 10 ml of lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 5 mM imidazole) through the column. The lysis buffer was collected and the gel sample was removed. The column was then washed with 20 ml wash buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 40 mM imidazole, 10% glycerol) and another gel sample was prepared in the same manner. Proteins were eluted by adding buffer with increasing imidazole concentration (wash buffer with imidazole at either 100 mM, 200 mM, or 500 mM). 3 ml of each buffer was applied and 3 ml each of the 100 mM and 200 mM imidazole eluates were collected. The eluate of 500 mM imidazole was collected as a 0.5 ml fraction. In addition, a gel sample was prepared from 10 μg of the resin after elution, and it was determined whether or not the protein remained bound. All samples were tested on 4-20% SDS / PAGE gels (Novex), followed by Coomassie staining or Western blot using a 1: 2000 dilution of anti-myc-HRP conjugated antibody (Invitrogen). The purified protein was stored at 4 ° C for immediate use and frozen at -80 ° C for storage.

  Uptake of VP22 fusion protein was detected by immunofluorescence. Cells were grown to approximately 50% of saturated cell concentration in 35 mm wells. The medium was then removed and replaced with 1 ml serum free medium. Approximately 10 μg of purified VP22 fusion protein eluted in a wash buffer containing 500 mM imidazole was added directly to the serum-free medium. Cells were incubated for 20 minutes at 37 ° C. and then washed with 3 × 2 ml PBS. The cells were then fixed and permeabilized in methanol for 5 minutes and prepared for immunofluorescence as previously described (see Invitrogen pVP22 / myc-His Vector, catalog number V484-1). .

Alternatively, uptake of VP22 fusion protein was detected by Western blot. This technique was used to detect the accumulation of VP22 fusion protein in the nucleus of PC12 cells and Jurkat T-cells. Approximately 5 × 10 ⁵ cell suspensions of each type were transferred to a 15 ml Falcon tube. The cells were collected by centrifugation at 500 g for 5 minutes and then resuspended in 10 ml of PBS. Cells were washed again in the same manner, then resuspended in 1 ml serum-free medium containing approximately 10 μg VP22 / GFP fusion protein and incubated at 37 ° C. for 15 minutes. Following incubation, the cells were washed twice by centrifugation and resuspended in 10 ml PBS as described above. Cells were harvested by re-centrifugation and 100 μl ice-cold lysis buffer (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM dithiothreitol (DTT), 1% Triton X- 100) and incubated on ice for 10 minutes. The lysate was centrifuged at 10000 g for 10 minutes. The supernatant containing soluble cytoplasmic protein was removed and 4X protein sample buffer was added to the supernatant. The pellet containing the cell nuclei was resuspended in 100 μl of 1X protein sample buffer. Samples were run on a 4-20% SDS / PAGE gel (Novex) and transferred to a nitrocellulose membrane. Western blots were probed with anti-myc-HRP antibody conjugate (Invitrogen).
Example 5
Activity of VP22 fusion protein in recipient cells: Functional test of VP22 / Rev fusion protein
HIV Rev protein is encoded by HIV genomic RNA and regulates RNA splicing. The Rev protein can bind to a transcript containing the Rev response element (RRE), allowing for nuclear export and subsequent translation of this transcript (VW Pollard et al., Ann. Rev. Micobiol. 52: 491- 532, 1998). In the absence of Rev, transcripts containing RRE are complexed with HIV spliceosome but not spliced. Instead, they remain in the nucleus and decompose.

  In the following experiments, Rev binding to RRE in transcripts was used to activate reporter gene expression. A reporter plasmid (pCAT / RRE) containing a CMV promoter and a CAT reporter gene separated by a spliced donor site was prepared. RRE was located 3 'to the CAT gene site. Thus, CAT expression in response to Rev can be used to demonstrate Rev activity in VP22 / Rev fusion proteins.

  CHO cells were transfected with pCAT / RRE and then treated with either VP22 / Rev fusion protein or VP22myc-His control fusion protein. Expression of the CAT reporter gene was tested by Western blot of protein samples prepared from treated cells using antibodies against CAT protein. Samples were also prepared from cells transfected with CMV-CAT positive control plasmid without RRE. Positive control expression could be detected. When cells were transfected with pCAT / RRE and then treated with VP22 / myc-His control fusion protein, no expression of CAT was detected. However, CAT expression could be detected when cells transfected with pCAT / RRE were treated with the VP22 / Rev fusion protein. When 5 times the amount of VP22 / Rev protein was added, a clear increase in the level of CAT protein was detected. These results indicate that VP22 can deliver a functional Rev protein to the nucleus leading to reporter gene expression.

In HIV-infected cells, the Rev protein can shuttle between the cytoplasm and nucleus. The distribution of Rev among these intracellular compartments depends on the nuclear export signal present in the protein. In order to determine whether the nuclear export signal is functioning in the VP22 / Rev fusion protein of the present invention, the distribution of VP22 / Rev protein was examined by immunofluorescence using an antibody against the myc epitope tag described above. By this method, VP22 / Rev fusion proteins are detected in the cytoplasm and nucleus of the cell, which indicates that Rev's fusion to VP22 does not interfere with the ability of Rev to distribute in both the cytoplasm and nucleus. Is shown.
Example 6
Delivery of one or more molecules into the cell to modify cellular processes The following experiments show the VP22 fusion protein containing the small GTPase, rhoA, which is involved in the polymerization of actin fibrils in mammalian cells as a fusion partner. Figure 2 shows a method for modifying cellular processes in a cell by delivery to the cell. Previous studies have shown that leaving Swiss 3T3 cells serum-free for 16 hours depolymerizes the actin fibrils involved in maintaining cell shape, leaving behind soluble actin monomers Yes. When serum is added, rapid repolymerization of actin occurs and microfibrils are restored. This effect was created by microinjection into cells of activated rhoA protein expressed and purified from E. coli (A. Hall, Science 279: 509-514, 1998).

  To test whether the VP22-rhoA fusion protein can produce a similar effect, the VP22-rhoA fusion protein was transformed into E. coli using the pCRT7 / VP22-1-TOPO® plasmid. From and purified. Swiss 3T3 cells were treated with the purified protein as follows. 3T3 cells were grown in 35 mm wells to approximately 50% of the saturated cell concentration. The medium was then removed and replaced with 1 ml serum free medium. Cells were incubated for an additional 20 hours at 37 ° C. Approximately 1 μg of purified VP22 / rhoA or VP22myc-His fusion protein was then applied to the cells. After 20 minutes, the cells were washed with 3 × 2 ml of PBS and then fixed in 4% formaldehyde for 5 minutes (from Invitrogen, β-galactosidase staining kit). Cells were washed again with 3 × 2 ml phosphate buffered saline (PBS), permeabilized with 0.1% Tween-20® detergent in PBS for 5 minutes, and then with 2 × 2 ml PBS. Washed again. After blocking the cells with 10% fetal bovine serum (FBS) in PBS for 30 minutes, FITC-conjugated phalloidin (Sigma P-5282) in PBS / 10% FBS was added to a final concentration of 0.1 μg / ml. Incubated for 30 minutes.

  Since phalloidin binds more strongly to polymerized actin than to depolymerized actin, repolymerized microfibers can be visualized. The cells were washed again with 2 × 2 ml PBS and then observed using an Olympus fluorescent microscope and FITC filter. The purified fusion protein was applied to 3T3 cells that did not receive serum. In cells treated with the VP22 / myc-His control fusion protein without prior serum, the actin fibrils cannot be detected and the cells do not give serum that has not been treated with any fusion protein Looked the same. In contrast, in cells treated with VP22-rhoA fusion protein for 20 minutes, actin microfibrils could be clearly detected by phalloidin binding. The distribution of actin microfilaments in cells treated with the VP22-rhoA fusion protein appeared similar to that seen in cells that had not been treated with either fusion protein or serum. These results indicate that VP22 can be used to deliver functional rhoA fusion proteins to cells.

Wild-type rhoA protein appears to stimulate the polymerization of actin fibrils from the cell membrane, whereas VP22 protein is normally transported to the cell nucleus. Since VP22 / rhoA can stimulate the polymerization of actin fibrils in a similar manner, the distribution of VP22 / rhoA protein was tested by immunofluorescence using an antibody against the myc epitope tag of VP22 / rhoA protein (Invitrogen) . Most of the VP22 / rhoA fusion protein could be detected in the cytoplasm of the recipient cells, and only a few proteins appeared to reach the nucleus. These studies indicate that the VP22 / rhoA protein is retained at the site of rhoA activity and does not completely translocate to the nucleus.
Example 7
Delivery of VP22 fusion proteins to specific cell compartments by modification of VP22 The following experiment demonstrates the use of VP22 fusion proteins to regulate the distribution of fusion partners within specific cell compartments. The HIV Rev protein (CM Troy et al., Neuroscience 16: 253-61, 1996) contains sufficient leucine-rich sequences to direct heterologous sequences to exit the nucleus and enter the cytoplasm. Furthermore, when the nuclear export signal (NES) is fused to a heterologous protein containing the canonical SV40 larger T antigen nuclear localization signal, the protein is distributed between the cytoplasm and the nuclear compartment (W. Wen et al., Cell 82: 463 -473, 1995). Similarly, the Rev protein contains both nuclear and nuclear export sequences and is found in both the cytoplasm and nuclear compartment (U. Fischer et al., Cell 82: 475-483, 1995).

  In this experiment, from VP22 / myc-His with 11 amino acids Rev Nes inserted between the VP22 ORF and myc epitope tag to test the ability to deliver translocation protein fusion partners to cell locations other than the cell nucleus. The resulting fusion protein was expressed in E. coli as described above, purified and applied to cultured cells.

The distribution of the fusion protein among the intracellular compartments in the cultured cells was examined by the immunofluorescence method described above. The distribution of the fusion protein was verified by Western blot analysis of cells treated as follows. A suspension of 5 × 10 ⁵ cells was transferred to a 15 ml Falcon tube. Cells were harvested by centrifugation at 500 g for 5 minutes and then resuspended in 10 ml PBS. The cells were washed again in the same manner, then resuspended in 1 ml of serum-free medium containing approximately 10 μg of VP22 / GFP fusion protein and incubated at 37 ° C. for 15 minutes. After incubation, the cells were washed twice as before by centrifugation and resuspension in 10 ml PBS. Cells are recovered by re-centrifugation and 100 μl ice cold lysis buffer (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM DTT, 1% Triton X-100® surfactant) And incubated for 10 minutes on ice. The lysate was centrifuged at 10000 g for 10 minutes. The supernatant containing the soluble cytoplasmic protein was removed and supplemented with 4X protein sample buffer. The pellet containing the cell nuclei was resuspended in 100 μl of 1X protein sample buffer. Samples were run on a 4-20% SDS / PAGE gel (Novex) and transferred to a nitrocellulose membrane. Western blots were probed with anti-myc-HRP antibody conjugate (Invitrogen). These studies show that Rev NES-conjugated-VP22-containing fusion protein can be distributed in the cytoplasm and nucleus of treated cells.
Example 8
Use of VP22 fusion proteins as components of inducible gene expression systems To test the theory that translocation proteins can be used in inducible gene expression systems with great specificity, what was described above for other VP22 fusion proteins The T7 RNAP / VP22 fusion protein was expressed from E. coli and purified using the same protocol as described above. RNA polymerase activity was tested by an in vitro transcription assay. All reagents were obtained from in vitro transcription kits (Ambion, Austin, TX) and used according to the manufacturer's instructions. The amount of RNA produced by the presence of the T7 RNAP / VP22 fusion protein was found to be similar to that of T7 RNAP included in the kit.

A reporter construct containing a luciferase gene driven by the T7 promoter was also constructed. This construct was transfected into COS cells and purified T7 RNAP / VP22 fusion protein 24 hours later was applied to the cells. After an additional 24 hours, cell lysates were prepared and tested for luciferase enzyme activity using a luciferase assay kit (Promega) according to the manufacturer's instructions. Addition of T7 RNAP / VP22 fusion protein to cells transfected with the reporter gene increases the level of luciferase expression to 5-10 times the background, indicating that the system is capable of expressing heterologous genes in eukaryotic cells. It is shown to function to control expression.
Example 9
Covalently and non-covalently coupled peptide or oligonucleotide molecules to the rearranged protein can be combined with Linx® chemical affinity using the low molecular weight chemical affinity ligands salicylhydroxamic acid (SHA) and phenylboronic acid (PBA). The system (Invitrogen) may be used to covalently conjugate to the translocated protein. In this system, a low molecular weight chemical affinity ligand is used to form a bifunctional linker that attaches the translocating protein to the polynucleotide by a reversible pH sensitive covalent bond.

Nucleic acid molecules containing PBA can be synthesized using PBA-NTPs (ProLinX, Seattle, WA) or, if double stranded, labeled with PBA-ATP using an enzyme terminal transferase. SHA-NHS esters can be used to attach SHA to lysine residues present in translocated proteins (FIGS. 3A-C). The PBA-conjugated molecule and SHA-conjugated translocation protein are then covalently linked and administered to the cell. A detailed description of the procedures and conditions used to ligate proteins using this system has been published (Linx ^™ Rapid Protein Conjugation Kit, catalog numbers K8050-01 to K8060-01, Invitrogen, San Diego, CA) .
Example 10
Translocation Protein: Oligonucleotide Conjugate Uptake Assay Translocation proteins and oligonucleotides of various lengths can be conjugated and added exogenously to mammalian tissue culture cells. Various lengths of single-stranded DNA (ssDNA) containing PBA-ATP can be synthesized using PCR. A biotinylated 5 ′ primer can be designed so that single-stranded molecules containing PBA-ATP can be purified on a streptavidin column. A series of 3 ′ reverse primers can be made to facilitate the synthesis of many ssDNA molecules 20 to 2000 nucleotides in length. Next, the purified ssDNA molecule containing PBA is mixed with translocation protein-SHA. Various concentrations of protein: oligonucleotide conjugates can then be added to the cells and incubated for up to 4 hours. Following incubation, the cells can be washed, fixed, and then probed with a strepavidin-FITC conjugate. Any incorporated oligonucleotide will bind to streptavidin-FITC and will be detected by fluorescence. Short oligonucleotides are expected to be taken up very efficiently (ie delivered to 100% of cells) and concentrated in the nucleus.

  Although the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention as set forth in the detailed description and claims.

FIG. 1 shows pFIN4 / lacZ having an intervening sequence (inv) adjacent to the Flp recognition site (flt) that separates the CMV promoter and β-galactosidase gene (lacZ). The interaction of Flp recombinase and pFIN4 removes the inv sequence and expresses β-galactosidase. Figures 2A-D deliver a fusion protein composed of VP22, anti-ATF-2 single chain antibody (sFv) and VP16 to the nucleus of the cell, which binds to ATF-2 and activates transcription It is a figure which shows the process to perform. FIG. 2A shows that the ATF-2 / LexA DNA binding domain (DBD) fusion protein binds to the minimal TK promoter and LexA operator (Op) upstream of the luciferase reporter gene but does not activate transcription. FIG. 2B shows that ATF-2 sFv-VP16 fusion protein binds to ATF-2 and activates transcription. FIG. 2C shows that the CREB sFv-VP16 fusion protein does not bind to ATF-2 and cannot activate transcription. FIG. 2D shows that a fusion protein composed of VP22, ATF-2 sFv and VP16 is delivered to the nucleus where it binds to ATF-2 and activates transcription. Figures 3A-C show the binding of the translocation protein (VP22) to the oligonucleotide (oligo) by the creation of a bifunctional linker molecule. FIG. 3A shows the chemical structure of phenylboronic acid (PBA) -conjugated nucleotide (PBA-dUTP). FIG. 3B shows the chemical structure of salicylhydroxamic acid (SHA) -conjugated amino acid (R = lysine). FIG. 3C shows the reaction of PBA-conjugated nucleotide and SHA-conjugated amino acid to create a bifunctional linker molecule that attaches the oligonucleotide to VP22. FIG. 4 is a diagram illustrating a VP22-T7 RNA polymerase (T7 pol) expression system. VP22-T7Pol accumulates in the nucleus upon exogenous addition to tissue culture cells. In the nucleus, the VP22-T7 pol fusion protein recognizes the T7 promoter and activates transcription of gene X. FIG. 5 is a map of the vector pVP22 / Myc-His, including the T7 promoter (T7), VP22 open reading frame (VP22), multiple cloning site, myc epitope (myc), and polyhistidine tag (6xHis). FIG. 6A shows the nucleotide sequence of vector pVP22 / Myc-His (SEQ ID NO: 1). FIG. 6B shows the nucleotide sequence of the vector pVP22 / Myc-His (SEQ ID NO: 1). FIG. 7 is a map of pVP22 / Myc-His-TOPO (registered trademark) vector, linearized vector of T7 promoter (T7), VP22 open reading frame (VP22), and vaccinia virus topoisomerase 1 protein (T). It contains a multicloning site modified by covalent attachment to DNA, a myc epitope (myc), and a polyhistidine tag (6xHis). A PCR product with a single 3′A base overhang can be inserted into the topoisomerase conjugation site. FIG. 8A shows the nucleotide sequence (SEQ ID NO: 2) of the pVP22 / Myc-His-TOPO® vector. FIG. 8B shows the nucleotide sequence (SEQ ID NO: 2) of the pVP22 / Myc-His-TOPO® vector.

Claims (30)

A method for regulating the expression of a target gene product in a cultured cell containing a target gene under the control of one or more regulatory elements, wherein the method comprises one or more conjugated to a translocating polypeptide under appropriate conditions. Control the expression of the target gene product by the cell by contacting the cultured cell with a regulatory substance and translocating the one or more regulatory substance into the cultured cell where it interacts with one or more regulatory elements The one or more regulators comprise a site-specific recombinase, and the cultured cell has a first nucleic acid having at least one site-specific genomic recombination site and a target gene and at least one site-specific recombination A second nucleic acid comprising a site, wherein the recombinase is recombination site specific and the translocation of the site specific recombinase is between the site specific recombination sites Cause e, so that the target gene is stably integrated into the cell to the genome in the genomic recombination site, method. The method of claim 1, wherein the site-specific recombinase is a family member of a site-specific recombinase selected from the group consisting of the integrase family of site-specific recombinases and the resolvase / invertase family of site-specific recombinases. The method according to claim 1, wherein the site-specific recombination site is a frt site and the site-specific recombinase is Flp. The method of claim 1, wherein the site-specific recombination site is a lox recombination site and the site-specific recombinase is Cre. A method for modulating the expression of a target gene product in a cultured cell containing a target gene under the control of one or more regulatory elements, wherein the method comprises one or more conjugated to a translocating polypeptide under appropriate conditions. Control the expression of the target gene product by the cell by contacting the cultured cell with a regulatory substance and translocating the one or more regulatory substance into the cultured cell where it interacts with one or more regulatory elements And wherein the one or more regulatory elements comprise a promoter and the one or more regulatory substances comprise a polymerase specific for said promoter. The method according to claim 5, wherein the polymerase is T7 RNA polymerase and the promoter is a T7 promoter. A method for regulating the expression of a target gene product in a cultured cell containing a target gene under the control of one or more regulatory elements, wherein the method comprises one or more conjugated to a translocating polypeptide under appropriate conditions. Control the expression of the target gene product by the cell by contacting the cultured cell with a regulatory substance and translocating the one or more regulatory substance into the cultured cell where it interacts with one or more regulatory elements One or more regulators comprise a DNA binding protein, wherein the DNA binding protein is a DNA binding domain of a steroid / thyroid hormone nuclear receptor superfamily member, a GAL4 DNA binding domain, a DNA binding domain of a homeobox protein , Zinc finger protein DNA binding domain, hormone receptor DNA binding domain, helix-turn-helix tan A method comprising a DNA binding domain selected from the group consisting of a DNA binding domain of a protein, a DNA binding protein of a basic Zip protein, a DNA binding protein of β-ribbon factor and a DNA binding domain of a Tet operon. 8. The method of claim 7, wherein the DNA binding domain has a DNA binding domain derived from a GAL4 DNA binding domain. The method according to claim 7, wherein the DNA binding domain is a domain represented by SEQ ID NO: 4. The method according to claim 7, wherein the DNA binding protein is a histone 1 (H1) protein or a non-histone protein HMG-17. 11. A method according to any one of claims 7 to 10, wherein the one or more regulatory elements comprise a promoter. A method for regulating the expression of a target gene product in a cultured cell containing a target gene under the control of one or more regulatory elements, wherein the method comprises one or more conjugated to a translocating polypeptide under appropriate conditions. Control the expression of the target gene product by the cell by contacting the cultured cell with a regulatory substance and translocating the one or more regulatory substance into the cultured cell where it interacts with one or more regulatory elements And wherein the one or more modulators comprise an HIV Rev protein and the one or more regulatory elements comprise an HIV Rev regulatory element (RRE). The method according to any one of claims 1 to 12, wherein the cultured cells are mammalian, yeast or insect cells. The method according to any one of claims 1 to 12, wherein the cultured cells are eukaryotic cells. 13. The method according to any one of claims 1 to 12, wherein the one or more modulators and the rearrangement polypeptide are linked non-covalently. The method according to any one of claims 1 to 12, wherein the translocating polypeptide and one or more regulatory substances are bound by a biotin-streptavidin complex or a single-stranded DNA binding protein derived from E. coli. 13. The method according to any one of claims 1 to 12, wherein the one or more modulators and the rearrangement polypeptide are covalently linked. 13. The method according to any one of claims 1 to 12, wherein the one or more modulators and the rearrangement polypeptide are linked by a linker. 19. The method of claim 18, wherein the linker comprises one or more disulfide bonds, salicylhydroxamic acid (SHA), phenylboronic acid (PBA), SHA-NHS ester, or combinations thereof. The method according to claim 18, wherein the linker is vaccinia DNA topoisomerase. 21. The method according to any one of claims 1 to 20, wherein the translocation polypeptide is a VP22 polypeptide, Antp or protein H. 21. A method according to any one of claims 1 to 20, wherein the translocation polypeptide is a VP22 polypeptide. 23. The method of any one of claims 1-14, 16-18 and 20-22, wherein the translocating polypeptide and the one or more modulators are units of a fusion protein. 24. The method according to any one of claims 1 to 23, wherein the target gene is further contained within a polynucleotide encoding a protein tag. 25. The method of claim 24, wherein the protein tag is a myc epitope, a fluorescent peptide or a poly His tag, or a combination of two or more thereof. The method according to any one of claims 1 to 24, wherein the target gene encodes a toxin protein. The method according to any one of claims 1 to 24, wherein the target gene is a reporter gene. 28. A vector for use in the method of any one of claims 1-27, comprising a polynucleotide encoding a cellular process modifying molecule bound to a translocating polypeptide. 29. The vector of claim 28, having the nucleotide sequence set forth in SEQ ID NO: 1. 29. The vector of claim 28, having the nucleotide sequence set forth in SEQ ID NO: 2.

Images