WO2000058488A2 - Delivery of functional protein sequences by translocating polypeptides - Google Patents
Delivery of functional protein sequences by translocating polypeptides Download PDFInfo
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- WO2000058488A2 WO2000058488A2 PCT/US2000/008571 US0008571W WO0058488A2 WO 2000058488 A2 WO2000058488 A2 WO 2000058488A2 US 0008571 W US0008571 W US 0008571W WO 0058488 A2 WO0058488 A2 WO 0058488A2
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
Definitions
- the present invention relates to methods for translocating polynucleotides and polypeptides between cells. More particularly, the present invention relates to use of translocating proteins to deliver a cell process-modifying molecule into the cell where the cell process-modifying molecule interacts specifically with a responsive target site.
- Translocating proteins are defined by their ability to cross biological membranes, such as cell membranes. A number of translocating proteins, have been described, including VP22 from Herpes Simplex Virus type 1 (G. Elliot and P. O'Hare, Cell fig, 223-233 (1997)), a fragment of the Nntennapedia protein from Drosophila (Antp) (D. Derossi et al., Journal of Biological Chemistry 269. 10444- 10450 (1994)), and Protein H from Streptococcus pyogenes (Axcrona et al., Manuscript in preparation (1999)).
- Antp 16 amino acid peptide corresponding to the third helix, named Antp, can translocate across membranes and accumulate in the cytoplasm and nucleus (Derossi et al, supra). This peptide is internalized at a temperature as low as 4°C, suggesting that endocytosis is not responsible for the internalization of the peptide. In addition, since translocation does not require classical endocytosis, Antp does not travel through the endosomal and lysosomal compartments. Therefore, Antp is resistant to proteolysis and has enhanced activity in most cellular compartments (D. Derossi et al., JBiol Chem 221:18188-18193, 1996).
- Antp as a vector peptide has been proven successful by genetically fusing 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 by covalent linkage via cysteine residues (D. Derossi et al., supra). Internalization of peptides as large as 41 amino acids and of charged phosphopeptides (B. Allinquant et al., J Cell Biol 12£: 19-927, 1995) has been demonstrated in neuronal cells.
- Antp is the only translocating peptide that has been used to deliver oligonucleotides (up to 45 nucleotides in length) to cells in culture (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 the human pathogen Streptococcus pyrogenes. Protein H is taken up by B- and T-lymphocytes and translocated to the nucleus. In contrast to other translocating proteins, which appear to have no effect on cellular function, protein H has a cytostatic effect thought to be the result of its association with the nuclear proteins SET and hnRNP A2 B1 (D. Derossi et al., supra). To date, the translocation of Protein H coupled to another molecule has not been demonstrated.
- the best studied of the translocating proteins is the Herpes Simplex Virus protein VP22, which has the unique ability to translocate between cultured mammalian cells.
- VP22 Herpes Simplex Virus protein
- the expressed protein accumulates in the cytoplasm of transfected cells and, by translocating across cell membranes, spreads to the surrounding non-transfected cells where it accumulates in the nuclei. This process can occur at 4°C and also appears to be energy-free and independent of endocytosis.
- export of VP22 can still occur.
- VP22- ⁇ 53 A. Phelan et al., Nature Biotechnology 16:440-443, 1998)
- VP22-thymidine kinase M.S. Dilber et al., Gene Therapy £: 12-21, 1999.
- At least twenty different mammalian cell types can take up a functional VP22-GFP fusion protein (Elliot and O'Hare, supra; Aints A., et al., J. Gene Med.
- Transfection of cells with plasmid DNA has been an invaluable tool for the study of biological systems.
- a variety of transfection methods e.g. lipids, calcium phosphate
- these methods rarely result in more than 50% of cells expressing a gene carried on a plasmid with which the cells are transfected. Since most cells do take up exogenous DNA, inefficient transfections do not appear to be due to inability of the DNA complex to enter the cell.
- the majority of DNA is internalized by endocytosis with very little of the internalized DNA ever reaching the cytoplasm or nucleus where expression takes place.
- the present invention overcomes these problems in the art by providing method(s) for modulating a cellular process in a cell in culture by contacting such a cell with a cell process-modifying molecule attached to a translocating polypeptide under suitable conditions, whereby the cell process-modifying molecule is translocated into the cells in culture and interacts specifically therein with a target site responsive to the cell process-modifying molecule, thereby modulating a cellular process in the cell.
- the present invention provides method(s) for transfecting a cell in culture with a target gene by contacting the cell under suitable conditions with a polynucleotide comprising the target gene attached to a translocating polypeptide, whereby the cell is transfected with the target gene.
- the present invention provides method(s) for modulating expression of a target gene product in a cell in culture that is transfected with the target gene under control of one or more regulatory elements by contacting the cell under suitable conditions with one or more regulatory agents attached to a translocating polypeptide, whereby the one or more regulatory agents are translocated into the cell and interact therein with the one or more regulatory elements, thereby modulating expression of the target gene product by the cell.
- the present invention provides vector(s) comprising a polynucleotide encoding a cell process-modifying molecule attached to a translocating polypeptide.
- FIG 1 is a schematic drawing showing pFIN4/lacZ, which has an intervening sequence (inv) flanked by Flp recognition sites (frt) separating the CMV promoter and ⁇ -galactosidase gene (lacZ). Interaction of Flp recombinase with pFIN4 results in the removal of the inv sequence and expression of ⁇ -galactosidase.
- Figures 2A-D are schematic representations of the process by which a fusion protein composed of VP22, an anti-ATF-2 single chain antibody (sFv), and VP16 is delivered to the nucleus of a cell where it binds ATF-2 and activates transcription.
- sFv anti-ATF-2 single chain antibody
- Figure 2 A shows the ATF-2/LexA DNA binding domain (DBD) fusion protein binds the LexA operator (Op) upstream of the minimal TK promoter and the luciferase reporter gene, but does not activate transcription.
- Figure 2B shows that the ATF-2 sFv-VP16 fusion protein binds ATF-2 and activates transcription.
- Figure 2C shows that the CREB sFv-VP16 fusion protein does not bind ATF-2 and cannot activate transcription.
- Figure 2D shows that the fusion protein composed of VP22, the ATF-2 sFv, and VP16 is delivered to the nucleus, where it binds ATF-2 and activates transcription.
- Figures 3A-C show the attachment of a translocating protein (VP22) to an oligonucleotide (oligo) by generation of a bifunctional linker molecule.
- Figure 3 A shows the chemical structure of a phenylboronic acid (PBA)-adapted nucleotide (PBA-dUTP).
- Figure 3C shows the reaction of the PBA- adapted nucleotide and the SHA-adapted amino acid to create a bifunctional linker molecule that attaches the oligonucleotide to VP22.
- PBA phenylboronic acid
- SHA salicylhydroxamic acid
- R lysine
- FIG. 4 is a schematic diagram illustrating a VP22-T7 RNA polymerase (T7 pol) expression system.
- VP22-T7Pol accumulates in the nucleus upon exogenous addition to tissue culture cells.
- the VP22-T7 pol fusion protein recognizes the T7 promoter and activates transcription of gene X.
- Figure 5 is a map of vector pVP22/ vc-His, which contains the T7 promoter (T7), VP22 open reading frame (VP22), a multiple cloning site, a myc epitope (myc), and a polyhistidine tag (6xHis).
- T7 T7 promoter
- VP22 open reading frame
- myc myc epitope
- 6xHis polyhistidine tag
- Figures 6A and B show the nucleotide sequence of vector pVP22/Myc-His (SEQ ID NO:l).
- Figure 7 is a map of pVP22/ yc-His-TOPO® vector, which contains the T7 promoter (T7), VP22 open reading frame (VP22), a multiple cloning site modified by covalent coupling of the Vaccinia Virus Topoisomerase I protein (T) to linearized vector DNA, a myc epitope (myc), and a polyhistidine tag (6xHis).
- T7 promoter T7 promoter
- VP22 open reading frame VP22
- T Vaccinia Virus Topoisomerase I protein
- myc myc epitope
- 6xHis polyhistidine tag
- Figures 8A and B show the nucleotide sequence of pVP22/Myc-His-TOPO® vector (SEQ ID NO:2).
- a cell process-modifying molecule attached to a translocating polypeptide, whereby the cell process-modifying molecule is translocated into the cell and interacts specifically therein with a target site responsive to the cell process-modifying molecule, thereby modulating a cellular process in the cell.
- translocating protein means a protein, polypeptide, or functional fragment thereof, that crosses biological membranes.
- Translocating proteins, polypeptides, functional fragments and homologues thereof possess the following properties: resistance to proteolysis, receptor-independent penetration of cell membranes, and substantially energy-free penetration of cell membranes.
- Exemplary translocating proteins that can be used in the invention methods and constructs include VP22 from Herpes Simplex Virus type 1 (G. Elliot and P.
- translocating proteins While each translocating protein has distinct properties, the general application of translocating proteins is to deliver other molecules to cells, either by constructing a fusion molecule (e.g., a fusion protein) or by attaching the desired molecule to the translocating protein (e.g. covalently or by means of a linker). In fusion proteins the translocating protein can be located either in the N-terminal or the C-terminal position.
- the preferred fusion protein or polypeptide for use in practice of the invention methods is a VP22 polypeptide.
- the term "VP22 polypeptide" is used herein to refer to the herpes viral VP22 protein, as well as to functional fragments thereof, that have the translocating properties of the intact protein.
- VP22 polypeptide encompasses homologues of VP22 protein, such as those derived from varicella zoster virus (VZV), equine herpesvirus (EHV), bovine herpesvirus (BHV), and the like, and transport-active (i.e. "functional") fragments, mutants and chimeric combinations thereof.
- VZV varicella zoster virus
- EHV equine herpesvirus
- BHV bovine herpesvirus
- VP22 polypeptide encompasses polypeptides corresponding to amino acids 60-301 and 159-301 of the full HSV1 VP22 sequence (1-301), whose sequence is disclosed in Figure 4 in WO 97/05265.
- Homologous proteins and fragments based on sequences of VP22 protein homologues from other herpes viruses are described in U.S. Patent 6,017,735, which is incorporated herein by reference in its entirety.
- fusion protein refers to two distinct proteins, polypeptides, peptides, and/or fragments not normally associated with each other in nature that are encoded by the same reading frame, resulting in the two or more distinct proteins and/or fragments being "fused” together.
- the fusion proteins used in invention methods are produced from nucleotide sequences encoding a translocating polypeptide, e.g., a VP 22 polypeptide, and another functional peptide in the same reading frame.
- polynucleotide encoding the fusion protein may also contain in the same reading frame additional peptide or polypeptide sequences useful in the invention methods, such as epitope-tag encoding sequences, affinity purification-tag encoding sequences, additional functional protein encoding sequences, and the like, or a combination of any two or more thereof.
- the invention provides method(s) for transfecting a cell with a target gene by contacting the cell under suitable conditions with a polynucleotide comprising the target gene attached to a translocating polypeptide, whereby the cell is transfected with the target gene.
- transfected means that a gene translocated into a cell in culture due to the translocating properties of an attached translocating polypeptide is expressed in the cell, at least transiently, i.e., the cell is transiently transfected with the target gene.
- the size of polynucleotide that can be transfected into a cell according to the invention methods ranges from about 10 nucleotides to about 10 kilobases (kb).
- polynucleotides in the range from about 20 nucleotides (nt) to about 5 kb, or from about 100 to 500 nt can be transfected into cells using the invention methods.
- the target polynucleotide is transiently transfected into a cell population in culture, for example, in a mono layer or tissue culture.
- translocating protein In invention methods wherein a translocating protein is attached to plasmid DNA (i.e., via either covalent or non-covalent interactions), the DNA can be delivered to the nucleus for gene expression. Delivery of DNA using translocating proteins as described herein is an extremely valuable research tool.
- a desired polynucleotide containing an open reading frame e.g., a polynucleotide contained in a plasmid
- the polynucleotide is internalized, transported to the nucleus, and the open reading frame is then expressed, thus creating a homogeneous population of cells for studying such cell processes as cell cycle regulation, transcription regulation, translation regulation, and the like.
- method(s) are provided for modulating expression of a target gene product in a cell in culture that contains a target gene under control of one or more regulatory elements.
- the invention method is practiced by contacting the cell in culture under suitable conditions with one or more regulatory agents attached to a translocating polypeptide, whereby the one or more regulatory agents are translocated into the cell in culture and interact therein with the one or more regulatory elements, thereby modulating expression of the target gene product by the cell.
- a polynucleotide attached to a translocating polypeptide can be translocated into the nucleus of the cell for expression of all or a part of the polynucleotide.
- the polynucleotide comprises an open reading frame encoding a protein of interest, such as a target gene product or reporter gene product.
- the polynucleotide can be a vector (e.g., a supercoiled plasmid) containing a cloned open reading frame that encodes a target gene.
- translocating protein and attached cell process- modifying molecule can be directed to the cytoplasm for expression as well as to the cell nucleus of the population of cells in culture if the translocating protein is attached (e.g., fused) to a nuclear export signal (NES).
- NES nuclear export signal
- incorporation of a NES is a potential method to modulate the nuclear targeting of translocating proteins, such as VP22, especially since the PKI NES can partially counteract the very strong signals of the SV40 NLS.
- the translocating polypeptide and any attached polynucleotide can be stably introduced into the cytoplasm as well as the nucleus of the cells in culture, thereby accomplishing partition of the polynucleotide between cellular compartments.
- regulation of expression of a gene contained in the polynucleotide can be regulated using invention methods as described herein.
- Nuclear export signals suitable for use in the practice of the invention are known in the art and include the nuclear export signals derived from the HIV Rev protein or the heat stable inhibitor of cAPK, and the like. In many cases, inclusion of a nuclear export signal into the translocation protein-containing construct can be used to stably integrate a target gene of interest into the genome of the cells in culture.
- a cell in culture can be contacted with a translocating protein attached to a cell-modifying molecule according to the present invention by a variety of methods.
- an expression cell population transfected with a polynucleotide encoding the translocating protein fused to the cell-modifying molecule e.g., as a fusion protein
- a target cell population that spontaneously takes up the expressed translocation protein with attached cell- modifying molecule.
- the expressed protein accumulates in the cytoplasm of the transfected expression cells and, by translocating across cell membranes, spreads to the surrounding non-transfected cells where it accumulates in the nuclei.
- the expression cell can be a prokaryotic cell line, such as E. coli
- the target cell line can be any eukaryotic cell line, for example a mammalian cell line, such as CHO or COS, or an insect cell line, such as Drosophila S2, and the like.
- the expression cell population can be cultured under conditions that promote expression of a transfected gene
- a cell lysate can be prepared of the transfected expression cell population and the lysate can be applied to a cultured target cell population using methods known in the art and as described in the
- the translocation protein is VP22
- the VP22 or fusion protein containing VP22 will translocate to the nuclei of substantially 100% of the cell population. It is also possible to culture the target cells with purified translocation protein-containing molecules or with synthetically prepared molecules containing the translocating protein attached to a polypeptide or nucleotide by means of a covalent bond or linker, as described herein.
- the translocating polypeptide and attached molecule will translocate to an entire cell population in culture within about 10 minutes to about 72 hours, more typically within about 10 minutes to about 50 hours; preferably within about 10 minutes to about 24 hours. However, in some cases, no more than about 10 minutes is required for uptake of a translocating polypeptide and attached molecule by an entire cell population.
- Fusions of translocating polypeptides with known DNA binding proteins can also be used to deliver DNA containing an open reading frame (e.g., a plasmid) to tissue culture cells.
- the DNA binding protein acts as a linker for attaching the translocating protein to the cell-modifying polynucleotide (i.e. a plasmid containing a polynucleotide that acts to modify a cell process).
- the cell-modifying polynucleotide i.e. a plasmid containing a polynucleotide that acts to modify a cell process.
- protein linkers that may be fused to translocating proteins for the delivery of polynucleotides, such as plasmid DNAs, include histone 1 (HI) protein (M. Wilke et al., supra and Niidome, et al., J. Biol. Chem.
- HMG-17 interactions with DNA have been studied in depth and demonstrate that HMG-17 interacts with DNA in a non-cooperative, nonspecific, and reversible manner (M. Bottger et al., Arch. Geschwulstforsch 6jQ, 265- 270 (1990)). In each case, either the entire DNA binding protein, or a functional fragment thereof (i.e. a fragment having DNA binding activity) may be used.
- a reagent such as polyethylenimine (PEI)
- PEI polyethylenimine
- the peptide linker may be fused to a translocating protein either as a chemically synthesized peptide or as a nucleotide encoding a fusion protein to be expressed in a prokaryote expression system.
- short peptide sequences that may be fused to a translocating protein either as a chemically synthesized peptide or as a fusion protein include polylysine sequences and sequences containing three or more repeats of the peptide sequence LARL, for example, LARL-LARL-LARL (SEQ ID NO:3) (J. D. Fritz et al., Hum. Gene Ther. 2:1395-1404 (1996)).
- LARL-LARL-LARL SEQ ID NO:3
- from three to about 100 repeats of the LARL sequence may be used as a linking peptide as described herein; typically from 3 to about 50 repeats, with 3 up to about 20 repeats being presently preferred.
- a preferred linker for attaching a translocating protein to a cell-modifying polynucleotide is the Vaccinia virus topoisomerase I protein, or a mutant form thereof, which allows the formation of stable topoisomerase I-DNA conjugates.
- Vaccinia DNA topoisomerase a 314 aa virus-encoded eukaryotic type I topoisomerase (I)
- binds to duplex DNA and cleaves the phosphodiester backbone of one strand S. Shuman and B. Moss, Proc. Nad. Acad. Sci. USA £4: 7478-7482 (1987)).
- the enzyme exhibits a high level of sequence specificity, akin to that of a restriction endonuclease.
- Vaccinia topoisomerase can religate the covalently held strand across the same bond originally cleaved (as occurs during DNA relaxation) or it can religate to a heterologous acceptor DNA and thereby create a recombinant molecule.
- the Vaccinia topoisomerase I linker When attached to an invention translocating protein, the Vaccinia topoisomerase I linker will attach to a double stranded oligonucleotide having single 5' A base overhangs, such as are created in Taq mediated PCR. Such topoisomerase I-DNA conjugates may then be introduced into cells.
- Figure 7 illustrates a suitable vector wherein Vaccinia topoisomerase I linker is used to attach a translocating protein to a double-stranded oligonucletide of interest.
- Vector pVP22 Myc-His TOPO® (SEQ ID NO:2), utilizes Vaccinia topoisomerase I linker to attach VP22 to a double stranded PCR product (i.e., a cell-process modifying oligonucleotide) having single 5' A base overhangs to create a VP22 fusion with vector DNA.
- a double stranded PCR product i.e., a cell-process modifying oligonucleotide
- Such topoisomerase I-DNA conjugates may then be introduced directly into cells.
- a translocating protein is used to increase the efficiency of plasmid delivery in conjunction with a cationic liposome.
- Fusion of a translocating protein to a protein domain that readily associates with a cationic liposome, for example a hydrophobic transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor facilitates interaction at the lipid-DNA interface.
- the translocating protein will translocate the complex through the endosomal membrane and into the cell cytoplasm and, eventually, to the nucleus for gene expression.
- Translocating proteins may also be used to enhance transfection efficiencies in conjunction with compounds, such as chloroquine, that inhibit lysosomal hydrolases (Niidome et al., J. Biol. Chem., 222:5207 -XI, 1998).
- Polynucleotides encoding fusion proteins may be constructed by standard molecular biology techniques (j. Sambrook, E. F. 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 translocation ability by use of suitable methods, e.g., immuno fluorescence, as are known in the art. See also the methods discribed in the Examples herein.
- Inducible systems are used to study the phenotypic effects of protein expression. Since inducible systems allow expression of a protein on demand, such systems can be used as a research tool to study cell processes and even to enable the expression of toxic proteins in tissue culture.
- Current systems for inducible mammalian expression use transcriptional elements from diverse organisms, for example, E.coli (U. Fischer et al., Cell £2:475-483 (1995)), or Drosophila (M. Gossen et al., 77BS1£:471-475 (1993)) that are constitutively expressed in a cell line along with a vector that contains a promoter responsive to transcriptional regulators.
- the present invention provides a novel approach to this problem by providing method(s) for modulating expression of a target gene product in a mammalian cell transfected with the target gene under control of one or more regulatory elements.
- the target cell is contacted under suitable conditions with one or more regulatory agents attached to a translocating polypeptide, whereby the one or more regulatory agents are translocated into the mammalian cell and interact therein with the one or more regulatory elements, thereby modulating expression of the target gene product by the cell.
- the translocating polypeptide used in invention methods for modulating expression of a target gene product can be any of the translocating polypeptides disclosed herein, but is preferably a VP22 polypeptide.
- the regulatory agent can be a polynucleotide, a protein or polypeptide, or a small molecule.
- the regulatory element can be a promoter operatively linked to a target gene wherein translocation of the regulatory agent transactivates expression of the target gene product by the promoter. It is preferred that the regulatory agent be specific for the promoter, such as a polymerase specific for the promoter.
- An exemplary inducible system utilizes the RNA polymerase of bacteriophage T7, which has been used to direct gene expression in mammalian cells.
- T7 RNA polymerase T7 RNA polymerase
- Vaccinia virus A. Ramsey-Ewing and B. Moss, J. Biol. Chem. 221:16962-16966, 1996; T. R. Fuerst et al., Proc. Natl. Acad. Sci. £_:8122-8126, 1986
- a stable cell line O. Elroy-Stein and B. Moss, Proc. Natl. Acad. Sci. £2:6743-6747, 1990 and A.
- T7 RNAP protein promotes specific expression of genes that are 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 that non-specific gene activation does not occur. Expression using T7 RNAP has been reported to be very strong, 6-fold higher than the RSV promoter in one case (A. Lieber et al., Nucleic Acids Res.
- T7 polymerase can be used to specifically regulate gene expression by the addition of a T7 RNAP/VP22 fusion protein to cells containing a T7 promoter construct.
- Direct delivery of T7 RNAP using VP22 technology allows specific control of gene expression and minimizes negative effects of delivery to non-target sites.
- the methods of the present invention allow for study of the phenotypic effects of protein expression on demand.
- toxic proteins Due to the specificity of the invention inducible system, the expression of toxic proteins can also be studied in tissue culture.
- Toxic proteins refers to proteins that have immediate intrinsic toxic potential for living systems, including those trans-dominant mutations in proteins leading to constituitively active forms of the protein. Thus toxic proteins are distinguished from "pro-drug” type molecules that require modification after expression to release a toxic potential.
- Non-limiting examples of toxic proteins that can be used in practice of the invention methods are various oncogene products, such as Raf and Ras oncogene products (Reviewed by Avruch et al. Trends in Biology 19:279-83, 1994).
- the regulatory agent can be a transcription factor specific for the regulatory element so that translocation of the regulatory agent transactivates expression of the target gene product.
- the translocating protein can be fused to a DNA binding domain, such as that from the Gal4 protein, and to a common transactivation domain, such as VP16 or B42.
- the translocating protein-containing fusion protein will localize to the nucleus and then specifically activate a promoter which contains upstream binding sites for the DNA binding domain incorporated into the fusion protein.
- DNA-binding protein(s) contemplated for use herein belong to the well-known class of proteins that are able to directly bind DNA and facilitate initiation or repression of transcription.
- Exemplary DNA-binding proteins contemplated for use herein include transcription control proteins (e.g., transcription factors and the like; 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, Ge «e l3_5_:83-92, 1993; W. M. Krajewska, Int. J. Biochem., 24:1885-1898, 1992.)
- transcription control proteins e.g., transcription factors and the like; see, for example, Conaway and Conaway, Transcription Mechanisms and Regulation, Raven Press Series on Molecular and Cellular Biology, Vol. 3, Raven Press,
- Transcription factors contemplated for use herein as a source of such DNA binding domains include, e.g., homeobox proteins, zinc finger proteins, hormone receptors, helix-turn-helix proteins, helix-loop-helix proteins, basic-Zip proteins (bZip), ⁇ -ribbon factors, and the like. See, for example, S. Harrison, "A Structural Taxonomy of DNA-binding Domains," Nature, 3_5_3_:715-719.
- Homeobox DNA-binding proteins suitable for use herein include, for example, HOX, STF-1 (Leonard et al., Mol. Endo., 2:1275-1283, 1993), Mat ⁇ -2, INV, and the like.
- the DNA-binding domain(s) used in the invention methods can also be obtained from a member of the steroid/thyroid hormone nuclear receptor superfamily, or be substantially the same as those obtained from a member of the superfamily.
- the DNA-binding domains of substantially all members of the steroidthyroid hormone nuclear receptor superfamily are related. Such domains consist of 66-68 amino acid residues, and possess about 20 invariant amino acid residues, including nine cysteines. Members of the superfamily are characterized as proteins which contain these 20 invariant amino acid residues.
- X designates non-conserved amino acids within the DNA-binding domain; an asterisk denotes the amino acid residues which are almost universally conserved, but for which variations have been found in some identified hormone receptors; and the residues enclosed in parenthesis are optional residues (thus, the DNA-binding domain is a minimum of 66 amino acids in length, but can contain several additional residues).
- Such DNA binding domains bind to 2-half site recognition sites, as is well known in the art to transactivate transcription under control of a response element comprising the recognition site.
- the GAL4 DNA binding domain does not interact with a 2-half site DNA recognition site.
- the DNA binding domain of the yeast GAL4 protein comprises at least the first 74 amino terminal amino acids thereof (see, for example, Keegan et al., Science 211:699-704, 1986).
- the first 90 or more amino terminal amino acids of the GAL4 protein will be 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.
- the tetracycline inducible system is well-known in the art (see, e.g, Gossen et al., Proc. Natl. Acad. Sci.£:5547-5551 (1992); Gossen et al., 77RS 1£:471_475 (1993); Furth et al., Proc. Natl. Acad. Sci. 21:9302-9306, (1994) ; and Shockett et al., Proc. Natl. Acad. Sci. 22:6522-6526 (1995)).
- Transcription modulating domains are of two types, those that activate transcription of a gene sequence operatively associated with a response element that is responsive to the invention system (i.e., transcription activation domains) and those that repress or de-activate transcription of a gene sequence operatively associated with a response element that is responsive to the invention system (i.e., transcription repression domains).
- transcription activation domains those that activate transcription of a gene sequence operatively associated with a response element that is responsive to the invention system
- transcription repression domains i.e., transcription repression domains
- the ability of the invention system to activate transcription of such a target gene is generally enhanced when the transcription modulating domain attached to the translocating protein is a transcription activation domain.
- Transcription activation domains contemplated for use in the practice of the present invention can be obtained from a variety of sources and are well 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 associated with a suitable DNA-binding domain.
- suitable activation domains can be obtained from the N-terminal region of members of the steroid/thyroid hormone nuclear receptor superfamily, from transcription factor activation domains, such as, for example, VP16, GAL4, NF-kB or BP64 activation domains, and the like (See, for example, M. Manteuffel-Cymborowska, Ada Biochim Pol. 46(11:77-89 (1999); T. Tagami et al., Biochem Biophys Res. Commun. 2i3i2 ⁇ :358-63 (1998), W. Westin, Adv Pharmacol, 47:89-112 (2000)).
- the activation domain presently preferred for use in the practice of the present invention is obtained from the C-terminal region of the VP 16 protein.
- Transcription repressor domains that can be used in the invention methods include those that repress transactivation of gene expression.
- Exemplary transcription repressor domains suitable for use as the transcription modulating domain in the invention methods include RAFT, CREM, MECP-2, SMRT, NcoR, mSin3A, RAR, TR, SMRTR, and the like.
- translocating proteins may be used in inducible expression systems for modulating expression of a target gene
- site-specific recombination sequences which are sequences of nucleic acids that are specifically recognized by a particular site- specific recombinase.
- Site specific recombinases are enzymes that catalyze the excision and/or recombination of nucleic acid sequences, and may form intermediate complexes with the transfer sequence DNA during the recombination event. These enzymes recognize a relatively short, unique nucleic acid sequence that serves as a site for both recognition and recombination.
- Recombinases particularly useful in the practice of the invention are those that function in a wide variety of cell types because such enzymes do not require any host specific factors and do not require ATP to function.
- telomeres Two major families of site-specific recombinases from bacteria and unicellular yeast have been described: the integrase family and the resolvase/invertase family.
- strand exchange catalyzed by site specific recombinases occurs in two steps of (1) cleavage and (2) rejoining, involving a covalent protein-DNA intermediate formed between the recombinase enzyme and the DNA strand(s).
- the nature of the catalytic amino acid residue of the enzyme and the line of entry of the nucleophile is different for these two recombinase families.
- the nucleophile hydroxyl is derived from a serine and the leaving group is the 3' -OH of the deoxyribose.
- the catalytic residue is a tyrosine and the leaving group is the 5' -OH.
- the rejoining step is the reverse of the cleavage step.
- Cre The recombinase activity of Cre has been studied as a model system for the integrases.
- Cre is a 38 kD protein isolated from bacteriophage PI . It catalyzes recombination at a 34 base pair stretch of nucleic acids called loxP.
- the loxP site has the sequence 5 '-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3 ' (SEQ ED NO: 5; spacer region underlined), consisting of two 13 base pair palindromic repeats flanking an eight basepair core sequence (Hoess et al., Proc. Natl. Acad. Sci USA 22:3398, 1982 and U. S. Patent No.
- the repeat sequences act as Cre binding sites with the crossover point occurring in the internal spacer core. Each repeat appears to bind one protein molecule wherein the DNA substrate (one strand) is cleaved and a protein-DNA intermediate is formed having a 3'-phosphotyrosine linkage between Cre and the cleaved DNA strand.
- the asymmetry of the core region of the loxP recombination sequence is responsible for directionality of the recombination reaction.
- Cre excises the DNA between these two sites, leaving a single loxP site on the DNA molecule (Abremski et al, Cell 12:1301, 1983).
- the repeat sequences act as Cre-specific binding sites with the recombination crossover point occurring in the core.
- the loxP site is so complex in size that it occurs only in the PI phage genome. Therefore, use of the loxP sites in the invention methods assures that the enzyme will not cut the transfer sequence within the interior of the sequence unless the transfer sequence is from the PI phage genome.
- mammalian cells including both rodent and human cells (van Deursen et al, Proc. Natl. Acad. Sci. USA 22:7376-7380, 1995; Agah et al, J. Clin. Invest. U ⁇ Q:169-179, 1997; Sauer and Henderson, New Biologist 2:441-449, 1990).
- the Cre protein also recognizes a number of variant or mutant lox sites (variant relative to the loxP sequence), including the loxB, loxL and loxR sites, which are found in the E. coli chromosome.
- Other variant lox sites include loxP511 t5'-ATAACTTCGTATAGTATACATTATACGAAGTTAT-3' (S ⁇ Q ID NO:6; spacer region underlined); loxC2
- loxP site can be prepared by those of skill in the art and will generally have no more than a total of one to three point mutations in the two repeats that comprise the site-specific recombination sequence.
- Cre catalyzes the cleavage of the lox site within the spacer region and creates a six base-pair staggered cut.
- the two 13 bp inverted repeat domains of the lox site represent binding sites for the Cre protein.
- the two lox sites may differ so long as Cre is able to recognize both lox sites.
- Cre cannot efficiently catalyze a recombination event using the two different lox sites.
- the efficiency of the recombination event will depend on the degree and the location of the variations in the binding sites.
- the loxC2 site can be efficiently recombined with the loxP site because the two lox sites differ by a single nucleotide in the left-binding site.
- Cre is the site specific recombinase used in the practice of the invention methods
- the site-specific recombination sequence is a loxP site, or a variant thereof recognized by the Cre enzyme.
- Flp a recombinase identified in strains of Saccharomyces cerevisiae that contain 2 ⁇ -circle DNA.
- Flp recognizes a DNA sequence consisting of two 13 basepair inverted repeats flanking an 8 basepair core sequence (5'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3 ' (SEQ ID NO: 8); spacer underlined) called FRT (Flp Recombination Target site).
- FRT Flp Recombination Target site
- the Flp gene has been cloned and expressed in E coli and in mammalian cells (PCT International Patent Application PCT/US92/01899, Publication No: WO 92/15694, the disclosure of which is herein incorporated by reference) and has been purified (Meyer-Lean et al, Nucleic Acids Res. 15:6469, 1987; Babineau et al, J. Biol Chem. 26J ⁇ :12313, 1985; Gronostajski and Sadowski, J. Biol. Chem. 26j :12328, 1985).
- Flp is functional in a wide variety of systems including bacteria (Huang et al, J. Bacteriology 122:6076-6083, 1997), insects (Golic and Lindquist, Cell 52:499-509, 1989; Golic and Golic, Genetics 144:1693-1711, 1996), plants (Lyznik et al, Nucleic Acids Res 21:969-975, 1993) and mammals (U. S. Patent Nos. 5,677,177 and 5,654,182), which shows the Flp does not require host specific factors for operability.
- Additional integrases that can be used in practice of invention methods are retroviral integrases, including HIV and ASV integrases (Reviewed in Annu. Rev. Microbiol. 53:245-81, 1990).
- a site-specific DNA recombinase or integrase fused to a translocating protein may be introduced as described herein into cells that have been transfected with a plasmid containing a transcription-blocking sequence (e.g., a transcription termination sequence) flanked by recombinase recombination sites specific for the recombinase or integrase used and placed between a promoter and an open reading frame encoding a target gene.
- a transcription-blocking sequence e.g., a transcription termination sequence
- the recombinase is Flp
- the recombinase sites are/rt sites
- the recombinase is Cre
- the recombinase sites are lox sites.
- the one or more regulatory elements can include a transcription-blocking sequence flanked by recombinase recombination sites and the regulatory agent can be a recombinase specific for the recombination sites, wherein translocation of the recombinase causes recombination of the recombination sites, thereby modulating expression of the target gene product.
- a single recombinase site can be incorporated into (or exist naturally in) the genome of the target cell that also contains a plasmid containing a target gene and a second recombinase site that pairs with the genomic recombinase site.
- a recombinase e.g.
- translocation of the recombinase will trigger a recombination event such that the target gene will become stably incorporated into the genome of the target cell at the genomic recombinase site.
- the recombinase or integrase may be introduced by mixing of two cell populations, one expressing the translocating protein-enzyme (e.g., Flp or Cre) fusion and the other containing the heterologous gene.
- the translocating protein-enzyme fusion may be produced in a prokaryotic or eukaryotic expression system, purified using known methods and as described herein, and applied to cells containing the heterologous gene.
- the cells may be either transiently transfected with the heterologous gene or carry it stably integrated in their genomes.
- the regulatory agent used in invention methods for modulating gene expression can be the HIV Rev protein paired with the Rev regulatory element as regulatory element (RRE). As illustrated in Example 5 herein, increasing amounts of Rev protein delivered to target cells containing RRE result in increased expression of an operatively linked target gene.
- RRE Rev regulatory element
- the protein molecule fused to a translocating protein is a Fv antibody fragment or a single chain antibody (sFv).
- sFv single chain antibody
- polynucleotide encoding a fusion protein containing the translocating protein and sFv is introduced into cells in culture, as described herein, for translocation to the cell nucleus and intracellular expression.
- the sFv is specific for an antigen target associated with intracellular machinery involved in a cellular function, for example, a target located within the cell nucleus, binding of the sFv to the intracellular target can interfere with cellular functions, such as Ras signaling (O. Elroy-Stein, T. R. Fuerst, B. Moss, Proc.
- the one or more regulatory agents used in the invention method(s) for regulating cell processes can include a Fv or sFv specific for a component of the one or more regulatory elements within the cells in culture (whether the regulatory element is native or transfected into the cells), wherein translocation of the Fv or sFv into the cell by the translocating protein and binding of the antibody to the component modulates expression of the target gene product.
- ATF-2 belongs to the bZIP family of transcription factors and controls gene expression via 8-bp ATF/CREB motifs, either as a homodimer or as a heterodimer — for instance, with Jun (S. Huguier et al., Molecular and Cellular Biology i£:7020-7029, 1998).
- the fusion protein is expressed within a reporter cell line that has ATF-2 bound upstream of a reporter gene, e.g., the bacterial luciferase gene
- a reporter gene e.g., the bacterial luciferase gene
- binding of the sFv in the fusion protein to the ATF-2 antigen in the cell nucleus triggers expression of the ATF-2 sFv-VP16 fusion ( Figure 2B), but not a CREB sFv-VP 16 fusion ( Figure 2C), resulting in expression of the reporter gene.
- This experiment demonstrates that the ATF-2 sFv is delivered to the cell nucleus where it binds the ATF-2 antigen.
- Fv as used herein means a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains but chemically linked;
- sFv as used herein means a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
- variable chains in a Fv may be noncovalent, as described in Inbar et al, Proc. Nat'l Acad. Sci. USA 6.2:2659-62, 1972.
- variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
- Exemplary linkers used to attach two segments of a Fv or to attach any other two proteins to can be a bifunctional cleavable cross-linker, such as ⁇ -succinimidyl (4-iodoacetyl)- aminobenzoate; sulfosuccinimydil (4-iodoacetyl)-aminobenzoate; 4-succinimidyl- oxycarbonyl- ⁇ -(2-pyridyldithio)toluene; sulfosuccinimidyl-6- [ ⁇ -methyl- ⁇ - (pyridyldithiol)-toluamido] hexanoate; N-succimmidyl-3-(-2-pyridyldithio)- proprionate; succinimidyl 6[3(-(-2-pyridyldithio)-proprion
- linkers can be attached to purified proteins using numerous protocols known in the art, such as those described in Examples 1 and 2 (see Pierce Chemicals "Solutions, Cross-linking of Proteins: Basic Concepts and Strategies," Seminar #12, Rockford, IL).
- the antibodies used in the invention methods are sFv, comprising V H and V L chains connected by a peptide linker.
- These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
- the linker in the sFv can be a peptide having from about 2 to about 60 amino acid residues, typically from about 5 to about 40, preferably from about 10 to about 30 amino acid residues.
- the linker moiety can be a flexible spacer amino acid sequence, such as those known in single- chain antibody research. Examples of such known linker moieties include GGGGS (SEQ ID NO:9), (GGGGS) n (SEQ. ID NO:10), GKSSGSGSESKS (SEQ ID NO:l l), GSTSGSGKSSEGKG (SEQ.
- a Diphtheria toxin trypsin sensitive linker having the sequence MGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO: 19) is also useful.
- the peptide linker moiety can be VM or AM, or have the structure described by the formula: AM(G 2 10 S) X AM wherein X is an integer from 1 to 11 (SEQ ID NO:20). Additional linking moieties are described, for example, in Huston et al, PNAS Little:5879-5883, 1988; Whitlow, M., et al, Protein Engineering 6_:989-995, 1993; Newton et al, Biochemistry 3 >:545-553, 1996; A. J. Cumber et al, Bioconj. Chem. 1:397-401, 1992; Ladurner et al, J. Mol. Biol.221:330-337, 1997; and U.S. Patent. No. 4,894,443, the latter of which is incorporated herein by reference in its entirety.
- the target gene within a cell in culture can be a reporter gene, such as is known in the art, for example, a non-endogenous gene encoding a detectable marker, such as the E. coli ⁇ -galactosidase gene, luciferase, or CAT.
- a reporter gene such as is known in the art, for example, a non-endogenous gene encoding a detectable marker, such as the E. coli ⁇ -galactosidase gene, luciferase, or CAT.
- polynucleotide that encodes the reporter gene an additional nucleotide sequence that encodes a protein tag, such as an antibody epitope (e.g., derived from Myc), a fluorescent peptide, or a poly His tag.
- a protein tag such as an antibody epitope (e.g., derived from Myc), a fluorescent peptide, or a poly His tag.
- translocating protein can be covalently conjugated to a translocating protein or to a polynucleotide encoding a translocating polypeptide for use in the invention methods using two low molecular weight chemical affinity ligands that can be attached to macromolecules like DNA and to proteins and which combine to form a linker useful in preparation of fusion proteins or fusion genes used in the invention methods.
- Two such low molecular weight chemical affinity ligands are salicylhydroxamic acid (SHA) and phenylboronic acid (PBA), which quickly react to form a reversible pH-sensitive covalent bond ( Figures 3 A-C), thus providing a convenient linker to attach a translocating protein to another protein or to a polynucleotide.
- nucleic acid molecules containing PBA can be synthesized using PBA-NTPs (available from ProLinx, Seattle, Washington) or, if double stranded, can be labeled with PBA- ATP using the enzyme terminal transferase.
- SHA-NHS ester can also be used to attach SHA to lysine residues present in a translocating protein.
- a PBA-adapted molecule and a SHA-adapted translocating protein are covalently linked and applied to cells.
- linkers such as disulfide bonds (which would be disrupted upon delivery to the cytoplasm) or bifunctional linker molecules (e.g., as disclosed herein) may also be used. Covalent linking using these or other linkers known in the art and disclosed herein provides a relatively stable attachment of the translocating protein to another molecule.
- Strepavidin binds biotin very strongly (the disassociation constant is approximately 10 "15 ).
- This strong affinity which is routinely used to attach proteins to substrates, can be used to form a linker that attaches a cell process-modifying molecule to a translocating polypeptide.
- a fusion protein containing VP22 translocating protein and strepavidin may be generated and complexed with a biotinylated oligonucleotide to form a linker attaching a cell process-modifying polynucleotide to a translocating polypeptide.
- Another polypeptide molecule that may be used as a linker to attach a cell process-modifying molecule to a translocating polypeptide for use in invention methods is the single stranded DNA binding protein (SSB) from E coli. Only 21 amino-acid residues (amino acid residues 2 through 22) of SSB appear to be involved in binding to ssDNA (i.e., "the functional fragment of SSB"). Furthermore, binding of SSB to ssDNA is not sequence specific. Therefore, a fusion protein containing a translocation protein fused to a functional fragment of SSB is an extremely attractive linker for attaching a translocating protein to an oligonucleotide or to plasmid DNA.
- SSB single stranded DNA binding protein
- linking molecules described above i.e., those containing PBA and SHA or strepavidin and biotin
- fusion proteins containing a SSB and a translocating protein can be attached to unmodified DNAs, thus providing time and cost-saving advantages.
- ORF open reading frame
- VP22/ yc-His Figure 5; SEQ ID NO:l
- the anti-myc epitope provides for easy detection of recombinant protein with Anti-myc antibody, and the poly His tag is useful for purification.
- the vector used was modified by covalent coupling of the Vaccinia Virus Topoisomerase I protein to linearized vector DNA (e.g., pVP22 TOPO® TA Cloning® Kit (Invitrogen)).
- linearized vector DNA e.g., pVP22 TOPO® TA Cloning® Kit (Invitrogen)
- the ORF of a gene product of interest i.e., a "fusion partner”
- a Topoisomerase-adapted vector encoding the VP22 polypeptide is pVP22/Myc-His TOPO® vector ( Figure 7; SEQ ID NO:2).
- the plasmid containing the VP22 gene fusion was then transfected into cells in culture
- COS or CHO cells were seeded into 6 well plates and grown to approximately 50% confluence prior to transfection.
- 5 ⁇ g DNA was diluted into 1.5 ml OptiMEM medium (Gibco BRL, Chagrin Falls, OH) and mixed with 15 ⁇ l Pfx-6 lipid (Invitrogen) for COS cells or 15 ⁇ l Pfx-7 lipid (Invifrogen) for CHO cells. Diluted DNA plus lipid was incubated with cells for 4 hr at 37 °C, then replaced with the appropriate medium and incubated for an additional 40-48 hr at 37°C.
- VP22 may be used to modulate expression of a functional gene product
- a system for delivery of the site specific DNA recombinase Flp was developed.
- COS cells expressing a VP22-Flp recombinase fusion protein were prepared as described above and mixed with CHO cells that had been transfected with a reporter plasmid pFIN4// ⁇ cZ ( Figure 1).
- pFIN4// ⁇ cZ Figure 1
- the reporter plasmid a segment of DNA that includes a transcriptional terminator, the Bovine Growth Hormone polyadenylation signal (Goodwin and Rottman, J. Biol. Chem.
- CHO cells transfected with pFIN4// ⁇ cZ only expressed ⁇ -galactosidase when mixed with COS cells that express the VP22-Flp fusion.
- Flp recombinase the segment of DNA containing the transcriptional terminator was removed by recombination of the frt sites, and ⁇ -galactosidase was expressed.
- Incubation of the population of CHO cells transfected with plasmid that expresses a VP22-GFP fusion protein, but does not express a VP22-Flp fusion protein did not result in expression of ⁇ -galactosidase.
- a cell free lysate was prepared from cells transfected with pVP22/mvc-His as follows: COS cells were grown to 50% confluence in a 100 mm dish (approximately 10° " cells). Cells were transfected with 20 ⁇ g of pVP22/mvc-His DNA using Pfx-6. Forty hours post-transfection, the cell monolayer was washed twice with PBS and then collected by scraping into 10 ml PBS. Cells were centrifuged at 500 g for 5 min and the PBS was aspirated from the cell pellet, which was then frozen on dry ice. Frozen cell pellets were stored at -80°C prior to preparation of lysates.
- the cell pellet was thawed on ice following addition of 0.5 ml ice cold lysis buffer (10 mM HEPES, pH 7.9, 400 raM NaCl, 0.1 mM ethylene diaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 5% glycerol).
- 0.5 ml ice cold lysis buffer (10 mM HEPES, pH 7.9, 400 raM NaCl, 0.1 mM ethylene diaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 5% glycerol.
- the entire supernatant was immediately added to 2 x 10 ⁇ cells in a 35 mm plate without removing the tissue culture media. After a 10 minute incubation at 37°C, the media was removed and VP22/myc-His protein located in the nuclei of the cells was detected by immunofluorescence as described above.
- An alternative method for the detection of VP22 fusion protein uptake in mammalian cells from a cell free lysate prepared from cells that express the fusion protein utilizes Western blot.
- HeLa, COS or CHO cells were plated at 50% confluence in 60 mm dishes. Following application of the lysate, the cells were washed once with PBS and then with PBS containing 500 mM NaCl to remove protein non-specifically bound to the outside of the cell. The cells were treated with trypsin for about 5 minutes to disassociate them from the plate and to digest any remaining extracellular peptide.
- the cells were solubilized and the proteins separated on a 4-20% Glycine gel (Invitrogen, Carlsbad, CA). The separated proteins were then transferred to nitrocellulose and probed with the appropriate antibody conjugated to horseradish peroxidase (HRP). The VP22 fusion proteins were then detected using chemiluminescence.
- HRP horseradish peroxidase
- the vector pCRT7/VP22-l was developed to allow expression and purification of VP22 fusion proteins from E. coli.
- This vector utilizes a C-terminal fragment of the VP22 protein (amino acids 159-301), which has proven sufficient for translocation of VP22 fusion proteins across cell membranes.
- VP22 fusion proteins were prepared containing various proteins as the fusion partner (including the HIV Rev protein and human protein rhoN), and the fusion proteins were expressed and purified . Activity of each fusion partner was demonstrated following uptake by cells in culture.
- pCRT7/VP22-l is derived from the pET9b vector backbone ( ⁇ ovagen, Madison, WI).
- sequence encoding the C- terminal region of VP22 sufficient for translocation activity amino acids 159 -301
- a fragment containing a multiple cloning site and myc and His tags from the vector pVP22/myc-His were inserted into the pET9b vector backbone.
- the multiple cloning site of pCRT7/VP22-l was derived from that of pVP22/myc-His.
- the pCRT7/VP22-l vector was prepared for coupling to Vaccinia Topoisomerase I in exactly the same way as in preparation of the pVP22/myc-His-TOPO® plasmid, as described above. Therefore, in this vector, the sequence encoding the ORF of a fusion partner can be either inserted into one of the multiple cloning sites or cloned as a PCR product into the topoisomerase cloning site in a way similar to that used with pVP22/myc-His or pVP22/myc-His-TOPO® plasmid.
- a VP22 fusion protein was expressed as follows. Ten ng pCRT7/VP22-l DNA was transformed into 50 ⁇ l BL21(DE3)plysS cells. The transformed cells were incubated at 37°C for 1 hour in 200 ⁇ l SOC medium, which was then diluted to 2 ml with Luria-Burtoni (LB) medium plus 50 ⁇ g/ml kanamycin and allowed to grow overnight at 37°C. The 2 ml culture was used to inoculate 50 ml LB medium containing 50 ⁇ g ml kanamycin.
- LB Luria-Burtoni
- the VP22 fusion protein was purified as follows: The cell pellet was thawed on ice 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: ⁇ -mercaptoethanol to 5 mM, ⁇ -toluenesulfonyl fluoride (PMSF) to 0.5 mM, leupeptin and pepstatin to 1 ⁇ g/ml each, and lysozyme to 1 mg/ml.
- ⁇ -mercaptoethanol to 5 mM
- PMSF ⁇ -toluenesulfonyl fluoride
- leupeptin and pepstatin to 1 ⁇ g/ml each
- lysozyme 1 mg/ml.
- the lysate was incubated on ice for 20 to 30 min and then sonicated for 3 x 10 sec while on ice. DNase and RNase were added to final concentrations of 10 ⁇ g/ml each. The lysate was left on ice for an additional 20 min, then drawn through a 21 gauge needle three times, and centrifuged at 20000 g for 15 min. Following centrifugation, a gel sample was prepared from the soluble supernatant. The supernatant was applied to a column containing 1 ml Probond resin equilibrated with lysis buffer (Probond beads interact with proteins tagged with 6 histidine arrays). The resin and supernatant were mixed in the column on ice for one to two hours. The column was then clamped vertically and the resin was allowed to settle.
- lysis buffer Probond beads interact with proteins tagged with 6 histidine arrays
- a sample of the supernatant was removed to test for the presence of unbound protein by SDS/PAGE.
- the resin was washed by allowing 10 ml lysis buffer (50 mM Sodium Phosphate pH 8.0, 300 mM NaCl, 5 mM imidazole) to pass through the column.
- the lysis buffer was collected and a 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 way.
- Protein was eluted by addition of buffers having increasing concentrations of imidazole (wash buffer with either 100 mM, 200 mM or 500 mM imidazole). 3 ml of each buffer was applied, and 3 ml of each of the 100 mM and 200 mM imidazole elutions were collected. The 500 mM imidazole elution was collected as 0.5 ml fractions. A gel sample was also prepared from 10 ⁇ g of the resin after elution to determine if the protein remained bound. All samples were examined on 4-20%
- Uptake of VP22 fusion proteins was detected by immunofluorescence.
- Cells were grown to approximately 50% confluence in 35 mm wells. The medium was then removed and replaced with 1 ml of serum free medium. Approximately 10 ⁇ g of the purified VP22 fusion protein, eluted in wash buffer containing 500 mM imidazole, was added directly to the serum-free medium. Cells were incubated at 37°C for 20 min and then washed with 3 x 2 ml PBS. Cells were then fixed and permeablized in methanol for 5 min and prepared for immunofluorscence as described previously (see Invitrogen pVP22/myc-His Vector, cat no. V484-1).
- uptake of VP22 fusion proteins was detected by Western blot. This technique was used to detect accumulation of VP22 fusion protein in the nuclei of PC 12 cells and Jurkat T-cells. Suspensions of approximately 5 x 10 5 cells of each type were transferred to 15 ml Falcon tubes. Cells were recovered by centrifugation at 500 g for 5 min and then resuspended in 10 ml PBS. Cells were washed again in the same way, then resuspended in 1 ml serum-free medium containing approximately 10 ⁇ g of the VP22/GFP fusion protein and incubated at 37°C for 15 min.
- the HIV Rev protein is encoded by HIV genomic RNA and is responsible for regulation of RNA splicing.
- the Rev protein can bind to transcripts that contain a Rev Response Element (RRE), allowing export of the transcript from the nucleus and subsequent translation (reviewed in V. W. Pollard et al., Ann. Rev. Micobiol. 52:491- 532, 1998).
- RRE Rev Response Element
- franscripts that contain RRE will complex with the HIV spliceosome, but are not spliced. Instead, they remain in the nucleus and are degraded.
- the binding of Rev to the RRE in a transcript was used to activate expression of a reporter gene.
- a reporter plasmid (pCAT/RRE) was prepared that contains a CMV promoter and a CAT reporter gene separated by a splice donor site.
- the RRE was located on the 3' side of the CAT gene site. Therefore, expression of CAT in response to Rev can be used to demonstrate the activity of Rev in the VP22/Rev fusion protein.
- CHO cells were transfected with pCAT/RRE and then treated with either VP22/Rev fusion protein or VP22/myc-His control fusion protein.
- Expression of the CAT reporter gene was examined by Western blotting of protein samples prepared from treated cells, using an antibody against the CAT protein.
- a sample was also prepared from cells fransfected with a CMV-CAT positive confrol plasmid that does not contain the RRE. Expression of the positive control could be detected.
- no expression of CAT could be detected.
- the Rev protein can shuttle between the cytoplasm and nucleus.
- the distribution of Rev between these intracellular compartments is dependent on a nuclear export signal present in the protein.
- the nuclear export signal functioned in the invention VP22/Rev fusion protein was examined by immunofluorescence using an antibody against the myc epitope tag, as described above.
- the following experiment demonstrates how a cellular process may be modified in a cell by delivery to the cell of a VP22 fusion protein that contains as the fusion partner the small GTPase, rhoA, which is involved in the polymerization of actin microfilaments in mammalian cells.
- rhoA small GTPase
- a VP22-rhoA fusion protein was expressed and purified from E. coli using pCRT7/VP22- 1 -TOPO® plasmid.
- Swiss 3T3 cells were treated with the purified protein as follows: 3T3 cells were grown to approximately 50% confluence in 35 mm wells. Then the medium was removed and replaced with 1 ml of serum free medium. Cells were incubated for an additional 20 hr at 37°C. Approximately 1 ⁇ g of either purified VP22/rhoA or VP22/myc-His fusion protein was then applied to the cells.
- Phalloidin binds to polymerized actin more strongly than to depolymerized actin, thus allowing for visualization of repolymerized microfilaments.
- Cells were washed again with 2 x 2 ml PBS prior to observation with an Olympus fluorescence microscope and FITC filter.
- the purified fusion protein was applied to serum-starved 3T3 cells. In cells that had been serum-starved and then treated with VP22/myc-His control fusion protein, no actin microfilaments could be detected and the cells appeared similar to serum-starved cells that had not been treated with either fusion protein.
- actin microfilaments could be clearly detected by binding of phalloidin.
- the wild type rhoA protein appears to stimulate polymerization of actin microfilaments from the cell membrane, but VP22 protein is normally transported to the cell nucleus. Since VP22/rhoA could stimulate the polymerization of actin microfilaments in a similar way, the distribution of VP22/rhoA protein was examined by immunofluorescence using an antibody against the myc epitope tag of the protein (Invitrogen). Most of the VP22/rhoA fusion protein could be detected in the cytoplasm of recipient cells and very little protein appeared to reach the nuclei. These studies show that VP22/rhoA protein may be retained at the sites of rhoA activity and not completely translocated to the nucleus.
- VP22 fusion protein Delivery of a VP22 fusion protein to a specific cellular compartment by modification of VP22.
- the following experiment demonsfrates use of VP22 fusion protein to regulate distribution of the fusion partner within a specific cellular compartment.
- the HIV Rev protein (C. M. Troy et al., Neuroscience 16_:253-61, 1996) contains a leucine rich sequence that is sufficient to direct heterologous sequences out of the nucleus and into the cytoplasm.
- fusion of the Nuclear Export Signal (NES) to a heterologous protein that includes the canonical SV40 larger T antigen Nuclear Localization Signal results in distribution of the protein between the cytoplasmic and nuclear compartments (W. Wen et al, Cell £2:463-473, 1995).
- the Rev protein contains sequences for both nuclear import and export and is found in both the cytoplasmic and nuclear compartments (U. Fischer et al., Cell £2:475-483, 1995).
- a fusion protein that consists of VP22/myc-His with the eleven amino-acid Rev NES inserted between the VP22 ORF and the myc epitope tag was expressed in E. coli, purified as described above, and applied to cells in culture.
- fusion protein Distribution of the fusion protein among the cellular compartments in the cells in culture was examined by immunofluorescence as described above. The disfribution of the fusion protein was verified by western blot analysis of treated cells, as follows: A suspension of 5 x 10 5 cells was transferred to 15 ml Falcon tubes. Cells were recovered by centrifugation at 500 g for 5 min and then resuspended in 10 ml PBS. Cells were washed again in the same way, then resuspended in 1 ml serum- free medium containing approximately 10 ⁇ g VP22/GFP fusion protein, and incubated at 37°C for 15 min.
- RNA polymerase activity was examined in an in vitro transcription assay. All reagents were from an in vitro transcription kit (Ambion, Austin, TX), and were used according to the manufacnirer'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 the T7 RNAP included in the kit.
- a reporter construct that contains a luciferase gene driven by a T7 promoter was also constructed. This construct was transfected into COS cells and 24 hours later purified T7 RNAP/VP22 fusion protein was applied to the cells. After an additional 24 hours, cell lysates were prepared and examined for luciferase enzyme activity using a luciferase assay kit (Promega) according to the manufacnirer's instructions. Addition of T7 RNAP/VP22 fusion protein to cells transfected with the reporter gene resulted in five- to ten-fold increase above background in the level of luciferase expression, indicating that this system functions to control the expression of heterologous genes in eukaryotic cells.
- Peptide or oligonucleotide molecules may be covalently conjugated to translocating proteins using Linx® chemical affinity system (Invitrogen) which uses low molecular weight chemical affinity ligands salicylhydroxamic acid (SHA) and phenylboronic acid (PBA).
- Linx® chemical affinity system Invitrogen
- SHA salicylhydroxamic acid
- PBA phenylboronic acid
- the low molecular weight chemical affinity ligands are used to form a bifunctional linker that attaches the translocating protein to a polynucleotide by means of a reversible pH-sensitive covalent bond.
- Nucleic acid molecules containing PBA can be synthesized using PB A-NTPs (ProLinx, Seattle, WA) or, if double sfranded, labeled with PBA- ATP using the enzyme terminal transferase.
- SHA-NHS ester can be used to attach SHA to lysine residues present in translocating proteins ( Figures 3 A-C).
- the PBA-adapted molecule and the SHA-adapted translocating protein are then covalently linked and administered to cells.
- a full description of the procedures and conditions used to link proteins using this system is publicly available (LinxTM Rapid Protein Conjugation Kit, Catalog Nos K8050-01 to K8060-01, invitrogen, San Diego, CA).
- a translocating protein and oligonucleotides of varying lengths can be conjugated and added exogenously to mammalian tissue culture cells.
- Single stranded DNA (ssDNA) of varying lengths containing PBA- ATP can be synthesized using PCR.
- a biotinylated 5' primer can be designed to allow purification of single- stranded molecules containing the PBA- ATP on a strepavidin column.
- a series of 3' reverse primers can be generated to facilitate the synthesis of a number of ssDNA molecules between 20 and 2000 nucleotides in length.
- the purified ssDNA molecules containing PBA will then be mixed with the translocating protein-SHA.
- oligonucleotide conjugate can then added to cells and allowed to incubate for up to 4 hours. After incubation, the cells can be washed, fixed, and then probed using a strepavidin-FITC conjugate. Any internalized oligonucleotides will bind the strepavidin-FITC and be detected by fluorescence. It is expected that short oligonucleotides will be internalized very efficiently (i.e. delivered to 100% of the cells) and be concentrated within the nucleus.
Abstract
Description
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AU40555/00A AU775001B2 (en) | 1999-03-31 | 2000-03-31 | Delivery of functional protein sequences by translocating polypeptides |
JP2000608767A JP2002539839A (en) | 1999-03-31 | 2000-03-31 | Delivery of functional protein sequences by translocating polypeptides |
CA002368998A CA2368998A1 (en) | 1999-03-31 | 2000-03-31 | Delivery of functional protein sequences by translocating polypeptides |
US09/937,837 US6773920B1 (en) | 1999-03-31 | 2000-03-31 | Delivery of functional protein sequences by translocating polypeptides |
US10/865,538 US20050208657A1 (en) | 1999-03-31 | 2004-06-09 | Delivery of functional protein sequences by translocating polypeptides |
AU2004220829A AU2004220829A1 (en) | 1999-03-31 | 2004-10-15 | Delivery of functional protein sequences by translocating polypeptides |
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EP1165819A2 (en) | 2002-01-02 |
AU775001B2 (en) | 2004-07-15 |
AU2004220829A1 (en) | 2004-11-18 |
AU4055500A (en) | 2000-10-16 |
CA2368998A1 (en) | 2000-10-05 |
JP2002539839A (en) | 2002-11-26 |
JP2005323619A (en) | 2005-11-24 |
WO2000058488A3 (en) | 2001-01-18 |
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