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Description  |
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FIELD OF INVENTION
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.
BACKGROUND OF THE INVENTION
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
88, 223-233 (1997)), a fragment of the Antennapedia 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)).
Antennapedia is a homeoprotein with a DNA binding domain composed of three alpha helices with a beta-turn separating helix 2 and 3. Experiments have demonstrated that a 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.degree. 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., J Biol Chem 271:18188-18193, 1996).
Recent experiments showing that a reverse helix (i.e. the reverse primary sequence) and a helix composed of D-enantiomers can transverse plasma membranes at 4.degree. C. suggest that internalization of Antp involves the formation of inverted
micelles in the phospholipid bilayer, making entry into cells receptor-independent and energy free (H. Hall et al., Current Biol 6:580-587, 1996).
The usefulness of 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 128:919-927, 1995) has
been demonstrated in neuronal cells. In each case, the sequences fused to Antp retained their expected biological functions. Furthermore, Antp is the only translocating peptide that has been used to deliver oligonucleotides (up to 45 nucleotides in
length) to cells in culture (C. M. Troy et al., J Neuroscience 16 253-61, 1996; G. Elliot et al., J Virol 72:6448-6455, 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. When cells are transfected with a plasmid encoding the VP22 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.degree. C. and also
appears to be energy-free and independent of endocytosis. When protein trafficking though the cell is blocked using Brefeldin A, export of VP22 can still occur. Studies of cytoskeletal elements during VP22 trafficking suggest that the actin
cytoskeleton may be involved in export or import of VP22 (Elliot and O'Hare, supra).
Delivery of several functional VP22 fusion proteins has been described, including VP22-p53 (A. Phelan et al., Nature Biotechnology 16:440-443, 1998)) and VP22-thymidine kinase (M. S. Dilber et al., Gene Therapy 6:12-21, 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. 1:275-9, 1999; and Wybranietz W. A. et al., J. Gene Med. 1:265-274, 1999), including mouse skeletal myoblasts that
are refractory to 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 the study of biological systems. A variety of transfection methods (e.g. lipids, calcium phosphate) exist in the marketplace; however, 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. Indeed, observations of directly injected lipid-DNA complexes suggest that movement from the endosomes to
the cytoplasm and nucleus is the most important limitation to successful transfections (J. H. Richardson et al., Proc. Natl. Acad. Sci.92:3137-3141, 1995). Consistent with this observation, peptides with membrane fusion activity, like the fusogenic
peptide of hemagglutinin (J. Zabner et al., Journal of Biological Chemistry 270:18997-9007, 1995), or a nuclear targeting sequence (M. Wilke et al., Gene Therapy 3, 1133-1142 (1996)) can increase transfection efficiencies in some cases.
Thus, there is a need in the art for new and better methods for modulating expression in cells of target genes and for transfection reagents and methods of their use to overcome the major blocks to expression of transfected genes, i.e.,
degradation in the endosomes and the inability of DNA to enter the cell nucleus.
BRIEF DESCRIPTION OF THE INVENTION
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.
In another embodiment, 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.
In still another embodiment, 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.
In yet another embodiment, the present invention provides vector(s) comprising a polynucleotide encoding a cell process-modifying molecule attached to a translocating polypeptide.
BRIEF DESCRIPTION OF THE FIGURES
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 .beta.-galactosidase gene (lacZ). Interaction of Flp recombinase with pFIN4 results
in the removal of the inv sequence and expression of .beta.-galactosidase.
FIGS. 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. FIG.
2A 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. FIG. 2B shows that the ATF-2 sFv-VP16 fusion protein
binds ATF-2 and activates transcription. FIG. 2C shows that the CREB sFv-VP16 fusion protein does not bind ATF-2 and cannot activate transcription. FIG. 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.
FIGS. 3A-C show the attachment of a translocating protein (VP22) to an oligonucleotide (oligo) by generation of a bifunctional linker molecule. FIG. 3A shows the chemical structure of a phenylboronic acid (PBA)-adapted nucleotide (PBA-dUTP).
FIG. 3B shows the chemical structure of a salicylhydroxamic acid (SHA)-adapted amino acid (R=lysine). FIG. 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.
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. 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 vector pVP22/Myc-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).
FIG. 6 is a map of pVP22/Myc-His-TOPO.RTM. 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). A PCR product with a single 3' A base overhang can be inserted into the topoisomerase-adapted site.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there are provided method(s) for modulating a cellular process by contacting a cell in culture under suitable conditions with 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.
As used herein, the term "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. O'Hare, 1997, supra), a fragment of the Antennapedia protein from Drosophila (Antp) (amino acids 43 through 58) (5'-RQIKIWFQNRRMKWKK-3') (SEQ ID NO:21) (Axcrona et al., supra 1999), Protein
H from Streptococcus pyogenes (D. Derossi et al., J. Biol. Chem., 271:18188-93, (1996)), and the like. 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. In addition, the term "VP22 polypeptide" as used
herein 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.
In particular, 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 FIG. 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. Pat. No. 6,017,735, which is incorporated herein by reference in its entirety.
The term "fusion protein" as used herein 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. The 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.
In one embodiment, 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. As used herein, the term "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). For example, 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. Generally, the target polynucleotide is transiently transfected into a cell population in culture, for example, in a monolayer or tissue culture. None of the
conventional means used to assist transfection or transduction is required, such as electroporation, infection employing viral vectors, calcium phosphate transfection, dextran sulfate transfection, lipofection, cytofection, particle bead bombardnent, and
the like. Instead all that is required is contact (i.e., co-culture) of the cell population to be transfected with purified translocation protein or with synthetically prepared translocating protein having a polynucleotide of interest attached thereto
by means of a covalent bond or linker molecule, as described herein. Any type of prokaryotic or eukaryotic cell in culture can be transfected using invention methods, for example, mammalian, yeast, insect or plant cells. However, it is presently
preferred that the cells in culture be a monolayer of mammalian or insect cells.
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. In up to 100% of the cells into which a desired polynucleotide containing an open reading frame (e.g., a polynucleotide contained in a plasmid) is delivered by an invention translocating
protein, 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.
In another embodiment according to the invention, 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. In this embodiment,
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.
For example, a polynucleotide attached to a translocating polypeptide, such as VP22, can be translocated into the nucleus of the cell for expression of all or a part of the polynucleotide. In one embodiment, the polynucleotide comprises an open
reading frame encoding a protein of interest, such as a target gene product or reporter gene product. Alternatively, the polynucleotide can be a vector (e.g., a supercoiled plasmid) containing a cloned open reading frame that encodes a target gene.
It has been discovered that the 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). Signals for the export of proteins from the nucleus have recently been described. Analysis of PKI (heat stable inhibitor of cAPK, cyclic AMP-dependent protein kinase A) (Y. Wang et al., Gene
Therapy 4, 432-441 (1997)) and the HIV Rev protein (W. Wen et al., Cell, 82, 463-473 (1995)) has revealed a leucine rich sequence that is sufficient to direct heterologous sequences out of the nucleus and into the cytoplasm. Furthermore, fusion of the
NES to a heterologous protein that includes the canonical SV40 larger 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, the Rev protein contains
sequences for both nuclear import and export and is found in both the cytoplasmic and nuclear compartments of cells (Wen, et al, supra). Thus, 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. When attached to a nuclear export signal, 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. In the cytoplasm, 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. In the one method, an expression cell population transfected with a polynucleotide
encoding the translocating protein fused to the cell-modifying molecule (e.g., as a fusion protein) is mixed and co-cultured with 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. For example, the
expression cell can be a prokaryotic cell line, such as E. coli, and 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.
Alternatively, 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 Examples herein. When 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. In this embodiment of the invention methods, 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). Examples of 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. 272, 15307-15312 (1997)) and the non-histone protein HMG-17 (high mobility group 17) (S. V.
Zaitsev, et al., Gene Ther. 4, 586-592 (1997)). HMG-17 interactions with DNA have been studied in depth and demonstrate 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 each case, either the entire DNA binding protein, or a functional fragment thereof (i.e. a fragment having DNA binding activity) may be used.
It may be preferred to complex the DNA with a reagent, such as polyethylenimine (PEI), that condenses and neutralizes the charged DNA prior to mixing with the translocating protein, or translocating protein-DNA binding domain fusion.
Alternatively, if a shorter peptide linker is advantageous in the particular system used, 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. Examples of 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. 7:1395-1404 (1996)). In some cases, 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. Natl. Acad. Sci. USA 84: 7478-7482 (1987)). The enzyme exhibits a
high level of sequence specificity, akin to that of a restriction endonuclease. Cleavage occurs at a consensus pentapyrimidine element 5'-(C/T)CCTT-3' in the scissile strand (S. Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699 (1994); J. M.
Clark, Nucleic Acids Res. 16: 9677-9686 (1988); and S. G. Morham and S. J. Shuman, Biol. Chem. 267: 15984-15992 (1992)). In the cleavage reaction, bond energy is conserved via the formation of a covalent adduct between the 3' phosphate of the incised
strand and a tyrosyl residue (Tyr-274) of the protein. 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. 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.
FIG. 6 illustrates a suitable vector wherein Vaccinia topoisomerase I linker is used to attach a translocating protein to a double-stranded oligonucleotide of interest. Vector pVP22/Myc-His TOPO.RTM. (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. Such topoisomerase I-DNA conjugates may then be introduced
directly into cells.
In another embodiment, 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. Following endocytosis of the liposome-DNA complex, 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., 272:15307-12, 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, N.Y.), transfected into tissue culture cells and tested for translocation ability by use of suitable methods, e.g., immunofluorescence, 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 82:475-483 (1995)), or Drosophila (M. Gossen et al.,
TIBS 18:471-475 (1993)) that are constitutively expressed in a cell line along with a vector that contains a promoter responsive to transcriptional regulators. Addition of an effector molecule causes binding of the transcriptional regulators to the
inducible promoter, thus turning on gene expression.
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. In
the invention method, 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 | | |
