WO2004056854A1 - Molecular transporters for intracellular delivery and their applications - Google Patents

Abstract

Peptide-based transport molecules capable of delivering a molecule of interest or cargo molecule into a eukaryotic cell, particularly the nucleus, are described. Also described are conjugates between the transport molecules and the molecules of interest which include GFP proteins, anti-sense nucleotides and small molecule inhibitors. Methods for the efficient cytoplasmic and nuclear delivery of biologically active proteins, nucleic acids and other molecules which are not inherently capable of entering target cells or cell nuclei or not inherently capable of entering cells or nuclei at a useful rate are accomplished by the use of the transport peptides which are described herein.

Description

MOLECULAR TRANSPORTERS FOR INTRACELLULAR DELIVERY

AND THEIR APPLICATIONS

FIELD OF THE INVENTION

The present invention relates to transport polypeptides, methods for making those transport polypeptides, transport polypeptide-cargo conjugates, pharmaceutical, prophylactic and diagnostic compositions comprising transport polypeptide-cargo conjugates, and methods for delivery of cargo into cells by means of transport polypeptides.

BACKGROUND OF THE INVENTION

Proteins, oligonucleotides and small organic molecules constitute a class of potential therapeutic agents. The bioavailability of drugs or molecular probes depends significantly on their polarities for administration, distribution and passive difϊusion. As a result, most drugs are limited to a narrow range of physical properties. In addition, many promising drug candidates fail to advance clinically because they fall out of this range, being either too nonpolar for administration and distribution or too polar for passive cellular entry. Several techniques have been developed to enable cellular uptake including drug incorporation into cationic liposomes (Rui, Y. J. et al, J. Am. Chem. Soc. 1998, 120, 11213-11218), dendrimers (Kono, K. et al., Biocoηjugate Chem. 1999, 10, 1115-1121), or siderophores (Ghosh, A. et al, Chem. Biol. 1996, 3, 1011-1019). Naturally occurring macromolecules enter into cells through an active transport mechanism. The nuclear transcription activator protein (Tat) encoded by HIV type 1 (HIV-1), a 101 amino acids protein that is required for viral replication (Lindgren, M. et al, Trends Pharmacol. Sci. 2000, 21, 99-103; Jeang, K.-T. et al, J. Biol. Chem. 1999, 274, 28837-28840) is one of the examples. By genetically or chemically hybridizing membrane permeable carrier peptides such as Tat from HIV-1 (48-60) and Antennapedia (43-58), the efficient intracellular delivery of various oligopeptides, peptide- oligonucleotide conjugates and proteins has been achieved (Futaki, S. et al, J. Biol. Chem. 2001, 276, 5836-5840; Wender, P. A. et al, Proc. Natl. Aca. Sci. 2000, 97, 13003-13008; Tung, C.-H. et al, Bioconjugate Chem. 2000, 11, 605-618; Schwarze, S. R. et al, Science 1999, 285, 1569-1572; Tomalia, D. A. et al, Drug Discovery Today 2001, 6, 427-436). The peptide mediated approaches would allow the incorporation of peptides containing unnatural amino acids or nonpeptide molecules such as peptoids and fluorescence probes. These methods would become powerful tools not only for the therapeutic purposes as an alternative to gene delivery, but also for the understanding of the mechanisms behind fundamental cellular events, such as signal transduction and gene transcription.

At the present time, the need exists for generally applicable means for safe, efficient delivery of biologically active molecules of interest or cargo molecules into the cytoplasm and nuclei of living cells.

SUMMARY OF THE INVENTION

In one embodimenζ the present invention relates to the discovery of transporter molecules based upon peptides to deliver a molecule of interest or cargo molecule into eukaryotic cells, particularly into the cell nucleus, in vitro or in vivo. In further embodiments it relates to conjugates between the transporter molecules and the cargo molecules where the cargo molecules may include GFP proteins, anti-sense nucleotides, small molecule inhibitors and other molecules of interest. The described conjugates are useful in the method of the present invention for delivering biologically active molecules into the cytoplasm and nuclei of cells.

In one embodiment, processes and products for the efficient cytoplasmic and nuclear delivery of biologically active proteins, nucleic acids and other molecules that are not inherently capable of entering target cells or cell nuclei, or not inherently capable of entering target cells at a useful rate are provided. Intracellular delivery of cargo molecules according to the present invention is accomplished by the use of novel transport proteins that comprise one or more cargo molecules and which are covalently attached to the cargo molecules. According to various embodiments, the present invention relates to novel transport polypeptides, methods for making those transport polypeptides, transport polypeptide-cargo conjugates, pharmaceutical, prophylactic and diagnostic compositions comprising transport polypeptide-cargo conjugates, and methods for delivery of cargo into cells by means of transport polypeptides.

In a preferred embodiment, the transport polypeptides are the nuclear localization sequence of Myo D (Vandromme, M. et al, Proc. Natl. Aca. Sci. 1995, 92, 4646-4650) and their modified sequences, the nuclear localization sequence of EGFR (Lin, S.-Y. et al, Nature Cell Biology 2001, 3, 802-808) and other nuclear localization sequences.

Transport polypeptides of the present invention may be advantageously attached to cargo molecules by chemical cross-linking or by genetic fusion. In one embodiment, a unique sulfhydiyl group is a preferred means of chemical cross-linking.

According to one preferred embodiment of the present invention, a biologically active cargo is delivered into the cells of various organs and tissues following introduction of a transport polypeptide-cargo conjugate into a live human or animal. By virtue of the foregoing features, the present invention opens the way for biological research and disease therapy involving proteins, nucleic acids and other molecules with cytoplasmic or nuclear sites of action.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows FACS and microscopic analyses for transduction. FACS analysis is shown in the upper view. A microscopic analysis is shown in the lower view for each transporter. Figure 1A, BMIP-134; Figure IB, B P-145; Figure 1C, BMIP-144; Figure ID, BMIP-139; Figure IE, BMIP-136; Figure IF, BMIP-141; Figure 1G, BMIP-140; Figure IH, BMIP-135; Figure II, BMIP-137; Figure 1J, BMIP-138.

Figure 2 shows the cell viability assay using SRB dye. Viability of treated cells was tested using the SRB assay. HeLa S3 cells were treated with varying concentrations of GOllll or R9 (250 nM to 50 μM) for 4 hr. Viability is retained in GOllll and R9 treated HeLa S3 cells.

Figure 3 A shows amino acid sequences of the three transporters: Tatrø.₅₇, BMIP-134 (EGFR₆₄₅-₆₅₇) and BMIP-146 (mMyoD). Mutated sequences in BMEP-146 (mMyoD) are underlined. Wild type sequence of MyoD is included for comparison. See body text for details.

Figure 3B. GFP or GFP-fusion transporters as indicated were analyzed by 16% SDS-PAGE and subsequently stained with Coomassie. Expression and purification of the proteins were as described below.

Figure 4. Dose- and time-dependent transduction of the peptide transporters. For dose-dependency, HeLa S3 cells were treated with 0.25, 0.5, 1, 2, or 4 μM each of the indicated transporters for 4 hrs at 37°C without serum (Figure 4A). For time-dependency, cells were treated with 1 μM each of the transporters and harvested at 1, 2, 4, 8 and 24 hr time points for analysis (Figure 4B). Transduction efficiencies were measured in FACS analysis using a FTTC channel of wavelength.

Figure 5. Image analyses for the transduction of the peptide transporters. HeLa cells were treated with the GFP or GFP-fused peptide transporters (4 μM each) as indicated for 4 hrs at 37°C without serum. Transduction of the transporters was assessed by the fluorescence image of GFP inside of the cells. HeLa cells were treated with all four proteins and GFP images were taken at the magnification of 200X (Figures 5A-D). Figure 5 A shows transport of GFP. Figure 5B shows

₆₅₇-GFP. Figure 5D shows transport of mMyoD-GFP. Images of BMIP-146-GFP (Figure 5E; mMyoD-GFP) and the corresponding morphology of HeLa S3 cells (Figure 5F) were taken at 400X to see the detailed localization pattern.

Figure 6. Cellular uptake of the peptide transporters in confocal images. HeLa cells were treated with 4 μM each of Tat₄₉.₅₇-GFP (Figure 6B), BMIP-134 (EGFR^s-rø-GFP) (Figure 6C) and BMIP-146 (mMyoD-GFP) (Figure 6D) for 4 hrs at 39°C without serum.

Figure 6A shows untreated. Fluorescent images were obtained using confocal microscope as described below.

Figure 7. Cytotoxicity of the peptide transporters. HeLa cells were treated with 1, 2, 4, or 8 μM each of the GFP or GFP-fusion transporters for 20 hrs as described. The viability of the cells was assessed using SRB assay. Tat ₉.₅₇-GFP and BMIP-134 (EGFR^s-es GFP) are described as Tat-GFP and EGFR-GFP, respectively in the legend for convenience.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In order that the invention herein described may be more fully understood, the following detailed description is set forth. In the description, the following terms are employed:

Amino acid

A monomeric unit of a peptide, polypeptide or protein. In general, the term refers to any compound having a carboxylic acid group, which also bears an amino group (-NH₂) on the carbon α to the carboxylic acid group. The twenty protein amino acids (L-isomers) include alanine ("Ala" or "A"), arginine ("Arg" or "R"), asparagines ("Asn" or "N"), aspartic acid ("Asp" or "D'), cysteine ("Cys" or "C"), glutamine ("Gin" or "Q"), glutamic acid ("Glu" or "E"), glycine ("Gly" or "G"), histidine ("His" or "H"), isoleucine ("lie" or 'T'), leucine ("Leu" or "L"), lysine ("Lys" or "K"), methionine ("Met" or "M"), phenylalanine ("Phe" or "F"), proline ("Pro" or "P"), serine ("Ser" or "S"), threonine ("Thr" or "T"), tiyptophan ("T " or "W"), tyrosine ("Tyr" or "Y') and valine ("Val" or "V"). The term amino acid, as used herein, also includes D-isomers of the above amino acids, synthetic amino acids, non-naturally occurring amino acids, and analogs of the protein amino acids.

Cargo

A molecule that is not a transport polypeptide or a fragment thereof, and that is either not inherently capable of entering target cells, or not inherently capable of entering target cells at a useful rate. Cargo, as used in this application, refers either to a molecule, per se, i.e., before conjugation, or to the cargo moiety of a transport polypeptide-cargo conjugate. Examples of cargo include, but are not limited to, small molecules and macromolecules, such as polypeptides, nucleic acids and proteins.

Chemical cross-linking

Covalent bonding of two or more pre-formed molecules.

Cargo conjugate

A molecule comprising at least one transport polypeptide moiety and at least one cargo moiety, formed either through genetic fusion, chemical cross-linking, or any other form of conjugation disclosed herein or known in the art, of a transport polypeptide and a cargo molecule.

Genetic fusion

Co-linear, covalent linkage of two or more polypeptides or proteins via their polypeptide backbones, through genetic expression of a DNA molecule encoding those proteins.

Macromolecule A molecule, such as a peptide, polypeptide, protein or nucleic acid.

Polypeptide

Any polymer consisting essentially of any amino acids, regardless of its size and sequence. Although protein is often used in reference to relatively large polypeptides, and peptide is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term polypeptide as used herein refers to peptides, polypeptides and proteins, unless otherwise noted.

Target cell

A cell into which a cargo is delivered by a transport polypeptide. A target cell may be any cell, including human cells, either in vivo or in vitro.

Transport moiety or transport polypeptide

A polypeptide capable of delivering a covalently attached cargo into a target cell.

Miscellaneous term

As used herein, "sequence identity" in the context of two polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to polypeptides it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide.

Certain embodiments of the present invention are based on the finding that when a polypeptide is present extracellularly, it is readily taken up into cells and subsequently into the cell nucleus. As a result of this finding, it is now possible to use specific polypeptides to deliver molecules such as proteins, peptides and nucleic acids into cells and, specifically, into the cell nucleus. Aspects of the present invention relate to methods of delivering a molecule of interest into cells and, particularly, of targeting a molecule to the cell nucleus, as well as a conjugate useful in the method. Any molecule can be delivered into cells, especially into the cell nucleus, using the methods of the present invention. For example, in one embodiment of the present method, the molecule to be delivered into cells is a protein, a peptide or an oligonucleotide. The present invention is particularly useful for delivery of proteins or peptides, such as regulatory factors, enzymes, antibodies, drugs or toxins, as well as DNA or RNA, into the cell nucleus.

I. CARGO CONJUGATE OF THE INVENTION

Thus, in the first aspect, the present invention relates to a transporter polypeptide molecule. The transporter polypeptide molecules of the present invention have the distinct characteristic of being endogenous to the host organism, or have a sequence that is substantially similar to the sequence of a polypeptide molecule found endogenously within a host organism. By "host organism" it is meant the organism that is to receive the polypeptide molecule. By "endogenous" it is meant that the polypeptide sequence is found naturally within a cell that is from the same order, preferably the same genus, and more preferably the same species, as the host organism, either by itself or as a part of a larger polypeptide or protein molecule.

Transporter polypeptide molecules are known in the art. See, for example, Futaki et al., "Arginine-Rich Peptides: An Abundant Source of Membrane-Permeable Peptides Having Potential as Carriers for Intracellular Protein Delivery," J. Biol. Chem. 276(6):5836- 5840 (2001), and references cited therein, which is hereby incorporated by reference herein in its entirety, including any drawings. However, these known transporter polypeptide are exogenous to mammals, i.e., they are obtained from non-mammalian cells, such as viruses. A problem faced with using exogenous polypeptide molecules is that the polypeptides may prove to be antigenic and initiate an immune response in the host organism. The use of endogenous polypeptide reduces, and likely eliminates, the antigenicity of the polypeptide molecule.

In certain embodiments, the polypeptide molecules of the invention are arg-nine-rich.

In other embodiments, the polypeptide molecules of the present invention are lysine-rich. By "arginine-rich" or "lysine-rich" it is meant that greater than 30% of the constituent amino acids of the polypeptide molecule of the invention is arginine or lysine, respectively.

In preferred embodiments, the polypeptide molecule of the present invention is selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 13, and preferably the group consisting of SEQ ID NO: 1 and SEQ ID NO:9 through SEQ ID NO: 13, and more preferably the group consisting of SEQ ID NO:9 through SEQ ID NO: 13. In other embodiments, the polypeptide molecule of the present invention has a sequence that comprises a sequence selected from the group consisting of SEQ ID NO:l through SEQ ID NO: 13, and preferably the group consisting of SEQ ID NO:l and SEQ ID NO:9 through

SEQ ID NO: 13, and more preferably the group consisting of SEQ ID NO:9 through SEQ ID

NO: 13. In still other embodiments, the polypeptide molecule of the present invention comprises an amino acid sequence having at least about 70%, preferably at least 80%, and more preferably at least about 90% identity to one of the amino acid sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 13. For example, the polypeptide molecule of the present invention comprises a sequence selected from the group consisting of SEQ ID NO:l through SEQ ID

NO: 13 that is missing at most 4, or at most 3, or at most 2, or at most 1 amino acid residues.

In another aspect, the present invention relates to a transporter polypeptide-cargo molecule conjugate comprising a transporter polypeptide as defined herein coupled to a cargo molecule. In some embodiments the cargo molecule is selected from the group consisting of proteins, polypeptides, nucleic acids, such as DNA or RNA, and organic molecules. In certain embodiments, the nucleic acid comprises an anti-sense nucleotide. In other embodiments, the cargo molecule comprises a polypeptide.

Organic molecules that can be used as cargoes within the scope of the present invention include, but are not limited to, hormones, vitamins, and therapeutic agent. Thus, in some embodiments, the organic molecule comprises a modulator of the function of a protein. A "modulator" is a compound that affects the function of a protein. In some cases, modulators increase the activity of a protein while in other cases, they decrease the activity of a protein. Modulators may be agonists or antagonists of a certain protein. The organic molecules as cargoes are those that bind, whether reversibly or irreversibly, to a protein. The protein may be a nuclear protein, a cytoplasmic protein, a membrane protein, or any other protein.

In certain aspects of the invention, the polypeptide transporter molecule of the present invention is coupled to the cargo. In some embodiments, the transporter molecule may be directly coupled to the cargo while in other embodiments a linker moiety may be placed between the transporter molecule and the cargo.

There are a number of ways by which the transporter molecule of the invention may be coupled to the cargo. In one embodiment, the transporter molecule is directly linked to the cargo through a covalent bond. In another embodiment, the transporter molecule may be coupled to the cargo using a cleavable linker, such as an acyl or an amide bond, which is capable of hydrolysis under cytoplasmic conditions, or is capable of hydrolysis by the action of an enzyme in the cytoplasm. In other embodiments, the linker may be susceptible to cleavage by the action of an enzyme, such as a glycosidase, protease, esterase, a redox enzyme, or a catalytic antibody.

In another embodiment, the transporter molecule is linked to another amino acid sequence which is the epitope tag ofan epitope on the surface of cargo. In this embodiment, the epitope tag binds to the epitope of the cargo much in the same way as an antibody binds to a peptidic moiety.

In another embodiment, the transporter molecule may be linked to a metal ion, which may be chelated by potential ligands on the cargo, or the cargo may comprise a metal ion, which may be chelated to potential ligands on the transporter molecule. In this embodiment, the ligating moiety, either the cargo or the transporter molecule, may comprise, for example, amino acid residues, such as histidines, or other residues, such as ethylenediaminetetraacetate, that will chelate to the metal atom on the metal-bearing moiety.

In another embodiment, the transporter molecule may be linked to a nucleic acid molecule which is complementary to a nucleic acid residue of the cargo. In this embodiment, the two complementary sequences hybridize to form the cargo conjugate.

In another embodiment, the transporter molecule may be linked to another polypeptide sequence, which is specific for a protease. The protease may be TEV protease, chymotrypsin, endoproteinase Arg-C, endoproteinase Asp-N, trypsin, Staphylococcus aureus protease, thermolysin, pepsin, or any other protease found in the organism into which the cargo conjugate is introduced.

In some embodiments, the transporter polypeptide is coupled to the cargo molecule by genetic fusion. In these embodiments, the genetic codes for the transporter polypeptide and the cargo molecule are put in the same vector, using standard molecular biology techniques. Once the vector is expressed, the transporter polypeptide couple to the cargo molecule is obtained. The transporter molecule in these embodiments may be directly attached to the cargo molecule, or there may be a polypeptide spacer between the transporter and the cargo molecules.

Thus, in another aspect, the present invention relates to a nucleic acid molecule which encodes the fusion polypeptide comprising the transporter molecule and the cargo molecule.

In some embodiments, the cargo conjugate also comprises a polypeptide linker between the cargo molecule and the transporter molecule. In other embodiments, the cargo conjugate also comprises a localization sequence, which allows for the cargo conjugate to be localized inside of a cell. In these embodiments, the liner sequence or the localization sequence, or both, are also part of the fusion polypeptide. Thus, the nucleic acid molecule of the present invention which encodes the fusion polypeptide encodes the fusion polypeptide in its entirety, including any linker or localization sequences that may be present.

In another aspect, the present invention relates to a vector comprising the nucleic acid molecule which encodes the fusion polypeptide comprising the transporter molecule and the cargo molecule. In yet another aspect, the present invention relates to a host cell comprising the above vector.

In another aspect, in addition to the cargo, transporter, and linker components thereof, a fourth component, a localization component, is added to the cargo conjugate. The localization component is a polypeptide molecule or an organic molecule that can direct where the cargo conjugate is taken up within the cell. Thus, for example, the localization component may be a nuclear localization sequence, which directs the cargo conjugate into the nucleus. It may be a substrate for a receptor on the endoplasmic reticular or mitochondrial membrane, which directs the cargo conjugate into the endoplasmic reticulum (ER) or the mitochondria, respectively. The localization component may also be a molecule that causes the cargo conjugate to be retained in the cytoplasm and not enter any of the cytoplasmic vesicles, such as ER or mitochondria or the nucleus. The same types of linkers discussed above, which can be used to link a transporter molecule to a cargo, can also be used to link a localization component to the cargo conjugate. However, it is understood that within the same molecule the transporter/cargo link may be different than the localization component/cargo conjugate link.

In another aspect, the cargo conjugate of the present invention could be implanted inside of an organism. In certain embodiments, the cargo conjugate will be in a solid, or liquid, formulation and kept inside of a pouch. The pouch may be fitted with a micro- electromechanical systems (MEMS) device. When the MEMS device is activated, for example, by applying an outside magnetic or electrical field, the pouch can then release a prescribed amount of the cargo conjugate into the blood stream of the organism.

π. SOME APPLICATIONS FOR THE CARGO CONJUGATE OF THE PRESENT INVENTION

In one embodiment, the disclosed transport peptides are applicable for therapeutic, prophylatic or diagnostic intracellular delivery of small molecules and macromolecules, such as proteins, nucleic acids and polysaccharides, that are not inherently capable of entering target cells at a useful rate. It should be appreciated, however, that alternate embodiments of the present invention are not limited to clinical applications. The present invention may be advantageously applied in medical and biological research. In some embodiments of the present invention, the cargo may be a drug or a reporter molecule. Transport polypeptides of the present invention may be used as research laboratory reagents, either alone or as part of a transport polypeptide conjugation kit.

A challenge faced with the treatment of neurological or psychotic disorders is that potential medications cannot easily pass through the blood-brain barrier (BBB). Under normal physiological conditions, a solute may gain access to brain interstitium via only one of two pathways: lipid mediation or catalyzed transport. Lipid-mediated transport is restricted to small molecules (with a molecular weight less than a threshold of approximately 700 Da) and is generally, but not always, proportional to the lipid solubility of the molecule. Catalyzed transport includes carrier-mediated or receptor-mediated processes. Thus, neither pathway is sufficient to allow easy administration of medications into the brain via the blood stream.

Of interest, is the administration of medications that can treat or ameliorate certain neurological disorders such as Alzheimer's disease, ALS, ataxia, autism, chronic pain, dystonia, epilepsy, Gaucher's disease, inclusion body myositis, multiple sclerosis, Parkinson's disease, stroke, syringomyelia, Tourette syndrome, and tremor. Also of interest is the administration of certain neurotropic factors, such as brain-derived neurotropic factor (BDNF), ciliaiy neurotropic factor (CNTF), glial-cell-line-derived neurotropic factor (GDNF), nerve growth factor (NGF), neurotrophin-3 (NT3), neurotrophin-4 (NT4).

The cargo conjugate of the present invention can readily pass the BBB and enter the neurons, thereby carrying the cargo component of the conjugate inside of the neuron. Employing the methods of the present invention, any of the medications used for the treatment of neurological disorders, or any of the factors enumerated above, or similar agents, can be considered a cargo and can be coupled to a suitable polypeptide transporter of the present invention to form a cargo conjugate. The cargo conjugate can then cross the BBB and deliver the cargo to neurons. Consequently, the dosage of the medication administered as a cargo conjugate would be significantly less than the dosage of the medication administered without the aid of the transporter molecules of the present invention. The lowering of the dosage greatly diminishes adverse side effects that can arise while treating neurological disorders.

Gene therapy, the introduction into the cell of a correct copy of a defective gene or the introduction into the cell of the gene for a polypeptide to be used for therapeutic purposes, is gaining wide-spread recognition as the therapeutic method of choice for the future. A seemingly insurmountable obstacle for gene therapy is the inability of the current methodologies for introducing the requisite DNA into the cells. Nucleic acids do not cross the cell membrane at an appreciable rate. Furthermore, the slow rate of DNA uptake results in DNA degradation prior to its entry into the cell or into the cell nucleus. The methods of the present invention allow for efficient delivery of DNA into cells or cell nuclei. The DNA can be coupled to one of the polypeptide transporter molecules of the present invention to form a cargo conjugate and administered to the patient. Another therapeutic method involving nucleic acids that has received generous attention recently is the use of antisense RNA molecules. Antisense RNA is complementary to the mRNA of a harmful protein. When antisense RNA is introduced into the cell, it hybridizes to the target mRNA, and as a result, prevents the mRNA to be translated. However, just as with gene therapy, antisense technology is being hampered by inadequate technologies to introduce the antisense molecule into the cell. The methods of the present invention allow for efficient delivery of the antisense molecule, coupled to a transporter polypeptide as a cargo conjugate, into cells or cell nuclei.

Ribozymes are RNA molecules that possess catalytic activity. The use of ribozymes for therapy has been suggested in the art. As with DNA and antisense RNA, the efficient introduction of ribozymes into cells has been hampered by their inability to cross the cell membrane at a useful rate. The cargo conjugates of the present invention comprising the ribozymes is an efficient method of introducing ribozymes into cells.

One of the biggest challenges facing researchers in the area of infection therapy is the alarming rate of increase in the antibiotic resistant strains of bacteria. One of the mechanisms by which bacteria become resistant to antibiotics is the development of efflux pumps that remove the antibiotic from the bacterial cytoplasm. However, it is conceived by the present inventors that when the antibiotics are introduced into the bacteria in the form of a cargo conjugate of the present invention, bacterial efflux pumps will not remove the antibiotics since the cargo conjugates of the present invention use a different transport mechanism than the one used by traditional delivery of antibiotics.

The use of silicon chips in conducting chemical and biological assays is becoming more prevalent. Already the art is familiar with chips comprising genes, polypeptides, and antibodies. However, cell-based assays using chips is very challenging since the art has not heretofore been able to find a way to successfully immobilize cells on a chip while allowing the cell to continue its normal biological function. The cargo conjugate of the present invention can be designed in such a way as to enable a cell to be immobilized on a chip without any loss or deterioration of its biological function. The cargo conjugate molecule can be linked to a tether, which may be another polypeptide molecule or a long chain organic molecule. The tether can then be attached to the chip. The cargo used in this conjugate would be a molecule that can bind to a cytoplasmic protein or component. For example, the cargo could be an antibody specific for a cytoplasmic protein. A cytoplasmic component or protein cannot cross the cell membrane and get out. The cargo conjugate then enters the cell, the cargo binds to the cytoplasmic site, while the transporter is linked to the tether and immobilized on the chip. Consequently, the cell becomes immobilized.

A chip can be designed such that at each location a cargo conjugate specific for a cell is tethered. Cells can then be added to the chip and be immobilized. Or the cargo conjugates used on a single chip can be specific for a single type of cells. Thus, chips can be obtained that comprise different cells at different locations or a single type of cell throughout. The cells thus immobilized retain their biological function. Cellular assays can then be performed on the chip.

EXAMPLES

Example 1 : Synthetic Polypeptides

Synthetic peptides were prepared with an automated peptide synthesizer (Applied Biosystems 433A) by using standard solid phase fluorenylmethoxycarbonyl (Fmoc) chemistry with HATU as the peptide-coupling reagent. The fluorescein moiety was attached by treating a resin bound peptide with carboxyfluorescein on the peptide synthesizer. Cleavage from the resin was achieved by using 95:5 trifluoroacetic acid (TFA)/triisopropylsilane. The solution from cleavage reaction was added dropwise to cold ether solution for the precipitation of synthesized peptide. The crude mixture thus obtained was centrifuged, the ether was removed by decantation, and the resulting orange solid was purified by reverse phase HPLC (water/acetonitrile in 0.1% trifluoroacetic acid). The products were isolated by lyophilization and characterized by electrospray mass spectrometry. The purity of the peptides was >95% as determined by analytical reverse HPLC (water/acetonitrile in 0.1% trifluoroacetic acid). The structures of the synthesized peptides are shown below in Table 1.

[TABLE 1]

Synthesized peptides for cellular uptake tests

BMIP-149 FL-DYDSEYESDSD (Poly Acidic Sequence: m)^(m) (SEQ ID NO: 13)

*NLS: Nuclear Localization Signaling Sequence

**(m): modified sequence a (Lin, S.-Y. etal, Nature Cell Biology 2001, 3, 802-808), b (Hall, M. N. etal, Cell 1984, 36, 1057-1065), c (Vandromme, M. et al, Proc. Natl. Acad. Sci. 1995, 92, 4646^650), d (Tinland, B. et al, Proc. Natl. Acad. Sci. 1992, 89, 7442-7446), e (Shoya, Y. etal, J. Virol. 1998, 72, 9755-9762), f (Shoya, Y. etal, J. Virol 1998, 72, 9755-9762), g (Prieve, M. G. etal, Mol. Cell Biol. 1998, 18, 4819-4832), h (Me, Y. et al, J. Biol. Chem. 2000, 275, 2647-2653), i (Vandromme, M. et al, Proc. Natl. Acad. Sci. 1995, 92, 4646-4650), j (Truant, R etal, Mol. Cell Biol. 1999, 19, 1210-1217), k (Vandromme, M. etal, Proc. Natl. Acad. Sci. 1995, 92, 4646-4650),

1 (Vandromme, M. etal, Proc. Natl. Acad. Sci. 1995, 92, 4646-4650), m (randomized polyacidic sequence).

Example 2 : Cellular Uptake Assay

Human epithelioid cervical carcinoma (HeLa S3) cells were treated with varying concentrations, from 1 to 50 μM, of each peptide, which was conjugated with fluorescein. The peptides were added directly to the media, and the delivery to the cells was visualized after 4 hr by microscopy.

Quantitation of the uptake of the peptide into the cells was performed by FACS analyses. Adherent cells, grown on 6 well plates, were displaced with trypsin, after which the cells were washed with phosphate-buffered saline (PBS). The final cell pellet was resuspended in PBS to a concentration of approximately 1 x 10 cells/mL. A minimum of 20,000 cell events were counted by flow cytometry. See Tables 2 and 3 below.

[TABLE 2]

Cellular uptake tests with transporter peptides

Cellular uptake efficiency (%):

A (0 - 10%), B (10 - 30%), C (30 - 60%), D (60 - 100%)

[TABLE 3]

Intracellular uptake efficiency

Cellular uptake efficiency (%): A (0 - 10%), B (10 - 30%), C (30 - 60%), D (60 - 100%) Example 3 : Cytotoxicity Assay for Transporter Peptide Molecules

Inhibition of cell growth was measured using a cell viability assay, which is based on the use of a protein-binding dye, sulforhodamine B (SRB). The different cancer cell lines were seeded in 96-well plates at 2000 cells/100 μL per well in RPMI media with 10% FBS. Next day, the cells were treated with compounds at designated doses and incubated for 72 hours at 37°C in the presence of 5% CO . Then the cells were fixed with 10% cold trichloroacetic acid (TCA) and incubated for an hour at 4°C. After washing the plates with tap water 4-5 times, the plates were dried in the air. Then the cells were stained with 0.4% SRB for 10 minutes, washed with 1% acetic acid 4-5 times and dried in the air. After adding 100 μL of 10 mM unbuffered Tris base to solubilize bound stain, optical densities were measured at 574 nm using Victor™ (1420 Multi-label Counter, Wallace) for the analysis. The values of ICso (the concentration of 50% growth inhibition) were calculated from the dose-response curve fitting. HeLa S3 cells were treated with varying concentrations of BMIP- 136 (NLS of MyoD) and BMIP-146 (mutated sequence of NLS of MyoD: mMyoD) (250 nM to 50 μM) for 4 hr. Viability is retained in BMIP-136 and BMIP-146 treated HeLa S3 cells even at 50 μM concentration of compounds (see Figure 2).

Example 4: Vectors of the peptide transporters fused with green fluorescent protein (GFP)

The bacterial expression vector for GFP with 6xHis tag (pET/GFP) was first constructed by inserting its coding region into the BamHI/Sall sites of pET21b plasmid

(Novagen, Inc.). Then three peptide transporters were fused in frame to the 5 '-end of GFP using BamHI/EcoRI sites in pET/GFP. The sequences of the peptide transporters are as follows: the basic domain of HIV Tat₄₉-₅₇ (RKKRRQRRR): 5'-cgtaagaaacgtcgtcagcgtcgtcgt-3';

the nuclear localization signal (NLS) of epidermal growth factor receptor (EGFR 64₅-₆₅7, RRRHIVRKRTLRR): 5-cgtcgccgtcatattgttcgtaaacgtaccctgcgtcgt-3';

the mutant form of NLS from a myogenic factor, BMIP-146 (mMyoD, VNLAFLTLRRC; the mutated sequences are underlined): 5'-gtgaacctggccttcctgaccctgcgtcgctgc-3'; and

the original amino acid sequence of BMIP-136 (MyoD) is VNEAFETLKRC.

The cloned plasmids were named as pET Tat ₉.₅₇-GFP, pET/EGFR₆₄₅-₆₅₇-GFP and pET/mMyoD-GFP, respectively.

Example 5: Preparation of peptide transporters with greeri fluorescent protein (GFP)

The plasmids for each transporter with GFP were introduced into a BL21(DE3) bacterial strain for expression. Cells were induced for expression of the proteins with 0.5 uM IPTG at 25°C overnight. The soluble proteins were first extracted from cells by sonication and subsequent centrifugation. Then they were affinity-purified using Ni-NTA agarose (Qiagen, Inc.) and removed of imidazole and salts using PD10 desalting column (Amersham Bioscience, Corp.) according to the manufacturer's instruction. The concentration of the proteins was determined using BCA assay (Pierce Biotechnology, Inc.).

Example 6: Transfer of the GFP-fused peptide transporters to animal cells

Human cervical carcinoma cell lines, HeLa and HeLa S3 cells were maintained in Dulbeco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and antibiotics. Cells were plated in 12- well plates the day before treatment. GFP-fused three peptide transporters were transferred to the cells in 500 μl of DMEM without serum. The concentration of the transporters and treatment time were as indicated in each set of experiment. Example 7: FACS and microscopic analyses for assessing transduction

After treatment, the transporters were removed and cells were washed with 1 ml PBS at least four times. Uptake of the transporters into cells was analyzed by FACS analysis or by microscopic observation of GFP fluorescence using a FITC channel of wavelength. In FACS analysis, a percentage of cells taken up GFP-fusion transporters was measured to the total number of cells counted. For confocal image analysis, cells were plated on the cover glass in 12-well plates and treated with 4 μM of each transporter for 4 hrs. After washing as above, cells were fixed with 3.7% para-formaldehyde for 10 min, counter-stained for actin and nucleus with Texas Red-X phalloidin (Molecular Probes, Inc.) and DAPI (Pierce Biotechnology, Inc.), respectively. Each process was performed at room temperature according to the manufacturer's instruction and followed by extensive washing with PBS. Finally, cover glasses were mounted on the slides and left in dark environment to dry. Fluorescence images were obtained at the magnification of 1000X in the confocal microscope using the wavelengths of 617 ran (actin), 528 nm (GFP) and 457 nm (nucleus). See Figure 1 A-J.

Example 8: Cell viability assay-sulphorhodamine B (SRB) assay

HeLa S3 cells were plated in a 96-well plate. The next day, the GFP-fused peptide transporters or GFP alone were treated to the cells at varying concentrations of 1, 2, 4 and 8 μM in a total volume of 100 μl media without serum. After 20 hrs of treatment, the cells were fixed by adding 25 μl of 50% trichloroacetic acid (TCA) for 1 hr at 4°C. Then cells were washed five times with distilled water and dried for a couple of minutes. Staining of cells was carried out using 100 μl of 0.4% SRB per well for 10 min at room temperature, followed by washing five times with 1% acetic acid. The staining was solubilized in 100 μl of 10 mM Tris buffer per well and measured at 572 ran (Figure 2). The indication of MyoD is the NLS of MyoD (BMIP-136), and the indication of mMyoD is the mutated sequence of NLS of MyoD (BMIP-146). Example 9: Design and preparation of the BMIP-134 fEGFR^g ) and BMIP-146 fmMvoD as GFP fusion protein

In an effort to find efficient molecular transporters, we selected two of the transporters, BMIP-134 (EGFR_M^) and BMIP-146 (mMyoD) (Fig. 3A). The BMIP-134 is a nuclear localization signals (NLS) from epidermal growth factor receptor (EGFR) and consists mostly of basic amino acids like a protein transduction domain (PTD) of HIV Tat-^ ₅₇. Especially enriched in both of the BMIP-134 and

are arginine residues, whose guanidinium head groups are suggested to be functional moieties in the transduction mechanism of Tatrø-₅₇. The BMIP-146 is a mutant form of the NLS from a member of helix- loop-helix myogenic factors, MyoD. Two acidic glutamates and a lysine residue in MyoD were changed to hydrophobic leucines and an arginine residue, respectively, which endowed it with both a hydrophobic nature and partially a property of argine. These peptide transporters were genetically fused to the N-terminus of green fluorescent protein (GFP) with a C-terminal His-tag and prepared using a bacterial expression system (Fig. 3B). The GFP alone was used as a negative control and Tat-^-^-GFP as a positive control, respectively.

Example 10: Transduction of the BMIP-134 EGFE s , and BMIP-146 (mMyoD) into animal cells

Time and dose-dependent transducing activities of the three peptide transporters were first assessed in HeLa S3 cells by FACS analysis as described above. When increasing amounts (0.25, 0.5, 1, 2 and 4 μM) of the GFP-fusion transporters were tested for 4 hrs (Fig. 4A), BMIP-134 showed quite a similar pattern of transduction efficiency to that of Tat₄₉-₅₇ which increased gradually from about 25% at 0.25 μM to near saturation at 4 μM. The BMIP-146 showed more efficient transduction than the other transporters starting from more than 50% transduction at 0.25 μM and reaching near saturation even at 2 μM of treatment. In the time-course experiment (1, 2, 4, 8 and 24 hrs) at 1 μM of these transporters, BMIP-134 showed again a similar pattern to Tat^ showing near saturation of transduction efficiency after 8 hr treatment starting from about 30% at 1 hr (Fig. 4B). About 60% of transduction efficiency was shown with luM of BMIP-146 even from 1 hr and it reached saturation after 4-8 hr of treatment. These results suggest that BMIP-146 is more potent than BMIP-134 and has a similar activity to a well-known PTD, Tat-_tø-₅₇ as molecular transporters.

Example 11: Cellular localization of the BMIP-134 (EGFR J . and BMIP-146 (mMyoD) in HeLa cells

The transduction of these peptide transporters was visualized by GFP images of the treated HeLa or HeLa S3 cells. When HeLa cells were treated with 4 μM of the transporters for 4 hrs, the transduction of BMIP-134 and BMIP-146 was as efficient as that of Tat-rø.₅₇ (Figure 5(b)-(d)). No transduction of GFP alone was observed (Figure 5(a)). The transduction activities were similar in HeLa S3 cells, but images were generally more comprehensive with HeLa cells due to their well-spread morphology (data not shown). Localization of Tat₄₉-₅₇- GFP at 4 μM was in the whole cell body with slight enrichment in the nucleus. The localization of BMIP-134-GFP was mainly in the nucleus at 4 μM with some staining in the cytoplasm. On the contrary, BMIP-146-GFP showed uneven distribution mostly in the cytoplasm but not in the nucleus of HeLa cells at 4 μM. The cytoplasmic/membrane localization of BMIP-146-GFP was more clearly seen in HeLa S3 cells thanks to their round- shaped and compact morphology (Figure 5(e)).

To address the localization of these transporters more clearly, confocal microscopic observation of the GFP fluorescence was performed in HeLa cells as described above. After treatment with 4 μM of transporters for 4 hrs, cells were fixed and counter-stained for actin (red) and nucleus (blue). As shown in Fig. 6, Tat-rø-₅₇-GFP localized both in the cytoplasm and in the nucleus as intense speckled forms (Figure 6(b)). By increasing the concentration of Tatrø-57-GFP up to 8 μM, its localization in the nucleus was increased (data not shown). The BMIP-134-GFP showed strong localization around the nucleus (Figure 6(c)), which is likely to be nuclear membranes because only weak and smearing signals were detected from the vertical scanning of focus for the GFP fluorescence (data not shown). This has been also suggested from the previous reports on the localization of EGFR Relatively weaker nuclear localization of Tat₄ -₅₇than that of BMIP-134 is probably because Tat₄₉.₅ is not an intact form of Tat NLS. Consistent with the result in Figure 5, the localization of BMIP-146-GFP was mostly in the cytoplasm as an irregular pattern and no nuclear staining was detected (Figure 6(d)).

Example 12: Cytotoxicity of the BMIP-134-GFP fEGFI s7-GFP) and BMIP-146-GFP faiMyoD-GFP)

To assess the cytotoxicity of the transporters, their effects on cell viability were examined. HeLa S3 cells in a 96 well plate were treated with various amounts (1, 2, 4 and 8 μM) of GFP or GFP fusion transporters in duplicate and the SRB assay was accomplished after 20 hrs as described above (Figure 7). The GFP alone showed slight toxicity to cells at all concentrations. The three transporters also showed only slight toxic effects until 4 μM concentration. Within this range, efficient transduction has been shown (See Figure 4). The transporters showed some toxicity at 8 μM, which, resulted in 85% (Tatj₉-₅₇-GFP), 80%

(BMIP-134-GFP) and 70% (BMIP-146-GFP) cell viability, respectively. However, 8 μM is far beyond the effective range both in terms of concentration and treatment time (Fig. 4).

Although Tat₄₉.₅₇ showed somewhat better results than BMIP-134 or BMIP-146, the toxicities of all three transporters were very similar. The toxicity difference at very high concentration (8 μM) could be from low levels of impurities in the purified proteins (not shown clearly in the gel image in Fig. 3B).

Taken together, these results suggest that our peptide transporters of BMIP-134 and BMIP-146 are equivalent to or better than the well-known PTD, Tatrø-5₇, in terms of transduction efficiency and toxicity.

As the present invention may be embodied in several forms without departing from the spirit or essential characteristics thereof, it should also be understood that the above- described examples are not limited by any of the details of the foregoing description, unless otherwise specified, but rather should be construed broadly within its spirit and scope as defined in the appended claims, and therefore all changes and modifications that fall within the meets and bounds of the claims, or equivalences of such meets and bounds are therefore intended to be embraced by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A transporter polypeptide comprising at least one amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO:l through SEQ ID NO: 13 and amino acid sequences having at least 70% identity to the amino acid sequences ofSEQ IDNO:l through SEQ IDNO:13.

2. The transporter polypeptide of claim 1 wherein the amino acid sequences are selected from the group consisting of amino acid sequences of SEQ ID NO:l and SEQ ID NO:9 through 13 and amino acid sequences having at least 70% identity thereto.

3. The transporter polypeptide of claim 2 wherein the amino acid sequences are selected from the group consisting of amino acid sequences of SEQ ID NO:9 through 13 and amino acid sequences having at least 70% identity thereto.

4. A transporter polypeptide-cargo molecule conjugate comprising the transporter polypeptide of claim 1 coupled to a cargo molecule.

5. The conjugate of claim 4 wherein the cargo molecule is selected from the group consisting of proteins, polypeptides, nucleic acids, and organic molecules.

6. The conjugate of claim 5 wherein the nucleic acid comprises an anti-sense nucleotide.

7. The conjugate of claim 5 wherein the cargo molecule comprises a polypeptide.

8. The conjugate of claim 5 wherein the organic molecule comprises a modulator of the function of a protein.

9. The conjugate of claim 4 wherein the transporter polypeptide is coupled to the cargo molecule by genetic fusion.

10. The conjugate of claim 4 wherein the transporter polypeptide is coupled to the cargo molecule by chemical cross-linking.

11. The conjugate of claim 10 wherein the chemical cross-linking is accomplished by using sulfhydryl groups.

12. The conjugate of claim 10 wherein the chemical cross-linking is cleavable.

13. The transporter polypeptide-cargo molecule conjugate of claim 4, wherein the transporter polypeptide comprises a peptide having the sequence of SEQ ID NO: 1.

14. The transporter polypeptide-cargo molecule conjugate of claim 4, wherein the transporter polypeptide comprises a peptide having the sequence of SEQ ID NO: 11.

15. A method of delivering a cargo molecule into a cell, the method comprising presenting to the cell a transporter polypeptide-cargo molecule conjugate, the conjugate comprising the transporter polypeptide of claim 1 coupled to the cargo molecule.

16. The method of claim 15 wherein the cargo molecule is selected from the group consisting of polypeptides, nucleic acids, and organic molecules.

17. The method of claim 16 wherein the nucleic acid comprises an anti-sense nucleotide.

18. The method of claim 16 wherein the cargo molecule comprises a polypeptide.

19. The method of claim 16 wherein the organic molecule comprises a modulator of a protein function.

20. The method of claim 15 wherein the transporter polypeptide is coupled to the cargo molecule by genetic fusion.

21. The method of claim 15 wherein the transporter polypeptide is coupled to the cargo molecule by chemical cross-linking.

22. The method of claim 21 wherein the chemical cross-linking is accomplished by using sulfhydryl groups.

23. The method of claim 21 wherein the chemical cross-linking is cleavable.

24. The method of claim 15 wherein the cell comprises a nucleus and the step of presenting the conjugate to the cell causes the cargo molecule to be delivered into the nucleus.

25. The method of claim 15, wherein the transporter polypeptide comprises a peptide having the sequence of SEQ ID NO: 1.

26. The method of claim 15, wherein the transporter polypeptide comprises a peptide having the sequence of SEQ ID NO: 11.

27. A pharmaceutical composition comprising the transporter polypeptide-cargo molecule conjugate of claim 4.

28. A nucleic acid molecule encoding the conjugate of Claim 9.

29. A vector comprising the nucleic acid molecule of Claim 28.

30. A host cell comprising the vector of Claim 29.