AU2001275423B2 - Encapsulation of polynucleotides and drugs into targeted liposomes - Google Patents

Description

WO 01/93836 PCT/US01/18657 ENCAPSULATION OF PLASMID DNA (LIPOGENES

T

AND

THERAPEUTIC AGENTS WITH NUCLEAR LOCALIZATION SIGNAL/FUSOGENIC PEPTIDE CONJUGATES INTO TARGETED LIPOSOME COMPLEXES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application Serial No. 60/210,925 filed June 9, 2000. The contents of this application is hereby incorporated by reference into the present disclosure.

FIELD OF THE INVENTION The present invention relates to the field of gene therapy and is specifically directed toward methods for producing peptide-lipid-polynucleotide complexes suitable for delivery ofpolynucleotides to a subject. The peptide-lipidpolynucleotide complexes so produced are useful in a subject for inhibiting the progression of neoplastic disease.

BACKGROUND OF THE INVENTION Throughout this application various publications, patents and published patent specifications are referenced by author and date or by an identifying patent number. Full bibliographical citations for the publications are provided immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Gene therapy is a newly emerging field of biomedical research that holds great promise for the treatment of both acute and chronic diseases and has the potential to bring a revolutionary era to molecular medicine. However, despite numerous preclinical and clinical studies, routine use of gene therapy for the treatment of human disease has not yet been perfected. It remains an important unmet need of gene therapy to create gene delivery systems that effectively target specific cells of interest in a subject while controlling harmful side effects.

WO 01/93836 PCT/US01/18657 Gene therapy is aimed at introducing therapeutically important genes into somatic cells of patients. Diseases already shown to be amenable to therapy with gene transfer in clinical trials include, cancer (melanoma, breast, lymphoma, head and neck, ovarian, colon, prostate, brain, chronic myelogenous leukemia, non-small cell lung, lung adenocarcinoma, colorectal, neuroblastoma, glioma, glioblastoma, astrocytoma, and others), AIDS, cystic fibrosis, adenosine deaminase deficiency, cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease, al-antitrypsin deficiency, rheumatoid arthritis and others.

Human diseases expected to be the object of clinical trials include hemophilia A and B, Parkinson's disease, ocular diseases, xeroderma pigmentosum, high blood pressure, obesity. ADA deficiency was the disease successfully treated by the first human "gene transfer" experiment conducted by Kenneth Culver in 1990. See, Culver, K.W. (1996) in: Gene Therapy: A Primer for Physicians, Second Ed., Mary Ann Liebert, Inc. Publ, New York, pp. 1-198.

The primary goals of gene therapy are to repair or replace mutated genes, regulate gene expression and signal transduction, manipulate the immune system, or target malignant and other cells for destruction. See, Anderson, W.F. (1992) Science 256:808-813; Lasic, D. (1997) in: Liposomes in Gene Delivery, CRC Press, pp. 1- 295; Boulikas, T. (1998) Gene Ther. Mol. Biol. 1:1-172; Martin, F. and Boulikas, T.

(1998) Gene Ther. Mol. Biol. 1:173-214; Ross, G. et al. (1996) Hum. Gene Ther.

7:1781-1790.

Human cancer presents a particular disease condition for which effective gene therapy methods would provide a particularly useful clinical benefit. Gene therapy concepts for treatment of such diseases include stimulation of immune responses as well as manipulation of a variety of alternative cellular functions that affect the malignant phenotype. Although many human tumors are non or weakly immunogenic, the immune system can be reinforced and instructed to eliminate cancer cells after transduction of a patient's cells ex vivo with the cytokine genes GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-y, and TNF-a, followed by cell vaccination of the patient intradermally) to potentiate T-lymphocyte-mediated antitumor effects (cancer immunotherapy). DNA vaccination with genes encoding tumor antigens and immunotherapy with synthetic tumor peptide vaccines are further WO 01/93836 PCT/US01/18657 developments that are currently being tested. The genes used for cancer gene therapy in human clinical trials include a number of tumor suppressor genes (p53, RB, BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc, K-ras), and suicide genes (HSV-tk, in combination with ganciclovir, cytosine deaminase in combination with 5-fluorocytosine). Other important genes that have been proposed for cancer gene therapy include bcl-2, MDR-1, p21, pl6, bax, bcl-xs, E2F, IGF-I, VEGF, angiostatin, CFTR, LDL-R, TGF-P, and leptin. One major hurdle preventing successful implementation of these gene therapies is the difficulty of efficiently delivering an effective dose ofpolynucleotides to the site of the tumor. Thus, gene delivery systems with enhanced transfection capabilities would be highly advantageous.

A number of different vector technologies and gene delivery methods have been proposed and tested for delivering genes in vivo, including viral vectors and various nucleic acid encapsulation techniques. Alternative viral delivery vehicles for genes include murine retroviruses, recombinant adenoviral vectors, adeno-associated virus, HSV, EBV, HIV vectors, and baculovirus. Nonviral gene delivery methods use cationic or neutral liposomes, direct injection ofplasmid DNA, and polymers.

Various strategies to enhance efficiency of gene transfer have been tested such as fusogenic peptides in combination with liposomes or polymers to enhance the release ofplasmid DNA from endosomes.

Each of the various gene delivery techniques has been found to possess different strengths and weaknesses. Recombinant retroviruses stably integrate into the chromosome but require host DNA synthesis to insert. Adenoviruses can infect non-dividing cells but cause immune reactions leading to the elimination of therapeutically transduced cells. Adeno-associated virus (AAV) is not pathogenic and does not elicit immune responses but new production strategies are required to obtain high AAV titers for preclinical and clinical studies. Wild-type AAVs integrate into chromosome 19, whereas recombinant AAVs are deprived of sitespecific integration and may also persist episomally.

Herpes Simplex Virus (HSV) vectors can infect non-replicating cells, such as neuronal cells, and has a high payload capacity for foreign DNA but inflict cytotoxic effects. It seems that each delivery system will be developed independently of the WO 01/93836 PCT/US01/18657 others and that each will demonstrate strengths and weaknesses for certain applications. At present, retroviruses are most commonly used in human clinical trials, followed by adenoviruses, cationic liposomes and AAV.

As the challenges of perfecting gene therapy techniques have become apparent, a variety of additional delivery systems have been proposed to circumvent the difficulties observed with standard technologies. For example, cell-based gene delivery using polymer-encapsulated syngeneic or allogeneic cells implanted into a tissue of a patient can be used to secrete therapeutic proteins. This method is being tested in trials for amyotrophic lateral sclerosis using the ciliary neurotrophic factor gene, and may be extended to Factor VIII and IX for hemophilia, interleukin genes, dopamine-secreting cells to treat Parkinson's disease, nerve growth factor for Alzheimer's disease and other diseases. Other techniques under development include, vectors with the Cre-LoxP recombinase system to rid transfected cells of undesirable viral DNA sequences, use of tissue-specific promoters to express a gene in a particular cell type, or use of ligands recognizing cell surface molecules to direct gene vehicles to a particular cell type.

Additional methods that have been proposed for improving the efficacy of gene therapy technologies include designing p5 3 "gene bombs" that explode into tumor cells, exploiting the HIV-1 virus to engineer vectors for gene transfer, combining viruses with polymers or cationic lipids to improve gene transfer, the attachment of nuclear localization signal peptides to oligonucleotides to direct genes to nuclei, and the development of molecular switch systems allowing genes to be turned on or off at will. Nevertheless, because of the wide range of disease conditions for which gene therapies are required, and the complexities of developing treatments for such diseases, there remains a need for improved techniques for performing gene therapy. The present invention provides methods and compositions for addressing these issues.

DISCLOSURE OF THE INVENTION A method is disclosed for encapsulating DNA and negatively charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers. The liposomes are able to reach primary tumors and their WO 01/93836 PCT/US01/18657 metastases after intravenous injection to animals and humans. The method includes micelle formation between DNA with a mixture of cationic lipid and peptide molecules at molar ratios to nearly neutralization ratios in 10-90% ethanol; the cationic peptides specify nuclear localization and have a hydrophobic moiety endowed with membrane fusion to improve entrance across the cell membrane of the complex. These peptides insert with their cationic portion directed toward condensed DNA and their hydrophobic chain buried together with the hydrophobic chains of the lipids in the micelle membrane monolayer. The DNA/lipid/peptide micelles are converted into liposomes by mixing with pre-made liposomes or lipids followed by dilution in aqueous solutions and dialysis to remove the ethanol and allow liposome formation and extrusion through membranes to a diameter below 160 nm entrapping and encapsulating DNA with a very high yield. The encapsulated DNA has a high therapeutic efficacy in eradicating a variety of solid human tumors including, but not limited to, breast carcinoma and prostate carcinoma. A plasmid is constructed with DNA carrying anticancer genes including, but not limited to p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-y, TNF-a, HSV-tk (in combination with ganciclovir), E. coli cytosine deaminase (in combination with 5-fluorocytosine) and is combined with encapsulated cisplatin or with other similarly systemically delivered antineoplastic drugs to suppress cancer.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the structure of the cancer targeted liposome complex.

FIG. 2 illustrates the results of plasmid DNA condensation with various agents as well as various formulation of cationic liposomes in affecting the level of expression of the reporter beta-galactosidase gene after transfection of K562 human erythroleukemia cell cultures.

FIG 3 illustrates tumor targeting in SCID mice. FIG 3A shows a SCID mouse with a large and small human breast tumor before and after staining with X-Gal to test the expression of the transferred gene. Both tumors turn dark blue. The intensity of the blue color is proportional to the expression of the beta-galactosidase gene.

WO 01/93836 PCT/US01/18657 FIG 3B shows that in the initial staining of the small tumor, the skin and the intestines at the injection area are the first organs to turn blue. FIG 3C is a view of the back of the animal. The two tumors are clearly visible after removal of the skin (top). Dark staining of the small tumor and light blue staining of the large tumor is evident at an initial stage of staining (bottom). FIG 3D is a view of the front side of the animal. The two tumors are clearly visible after removal of the skin. On the figure to the bottom the dark staining of both tumors is evident at a later stage during staining.

FIG 3E shows the front (top) and rear (bottom) higher magnification view of the dark staining of both tumors at a later stage during staining. Staining of the vascular system around the small tumor can also be seen (bottom).

BRIEF DESCRIPTION OF THE TABLES Table 1 is a list of molecules able to form micelles.

Table 2 lists several fusogenic peptides and describes their properties, along with a reference.

Table 3 lists simple Nuclear Localization Signal (NLS) peptides.

Table 4 shows a list of "bipartite" or "split" NLS peptides.

Table 5 lists "nonpositive NLS" peptides lacking clusters of arginines/lysines.

Table 6 lists peptides with nucleolar localization signals (NoLS).

Table 7 lists peptides having karyophilic clusters on non-membrane protein kinases.

Table 8 lists peptide nuclear localization signals on DNA repair proteins.

Table 9 lists NLS peptides in transcription factors.

Table 10 lists NLS peptides in other nuclear proteins.

MODES FOR CARRYING OUT THE INVENTION Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following WO 01/93836 PCT/US01/18657 publications. See, Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2 n d Edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F.M.

Ausubel, et al. eds., (1987); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR: A PRACTICAL APPROACH, M. MacPherson, et al., IRL Press at Oxford University Press (1991); PCR 2: A PRACTICAL APPROACH, MacPherson et al., eds.

(1995); ANTIBODIES, A LABORATORY MANUAL, Harlow and Lane, eds. (1988); and ANIMAL CELL CULTURE, R.I. Freshney, ed. (1987).

As used in the specification and claims, the singular form "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of' when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.

"Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention.

Embodiments defined by each of these transition terms are within the scope of this invention.

The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms ofnucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules.

WO 01/93836 PCT/US01/18657 A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

A "gene product" refers to the amino acid peptide or polypeptide) generated when a gene is transcribed and translated.

The following abbreviations are used herein: DDAB: dimethyldioctadecyl ammonium bromide (same as N,N-distearyl-N,N-dimethylammonium bromide); DODAC: N,N-dioleyl-N,N-dimethylammonium chloride; DODAP: 1,2-dioleoyl-3dimethylammonium propane; DMRIE: N-[l-(2,3-dimyristyloxy)propyl]-N,Ndimethyl-N-(2-hydroxyethyl) ammonium bromide; DMTAP: 1,2-dimyristoyl-3trimethylammonium propane; DOGS: Dioctadecylamidoglycylspermine; DOTAP (same as DOTMA): N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOSPA: N-(1-(2,3-dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate; DPTAP: 1,2- dipalmitoyl-3-trimethylammonium propane; DSTAP: 1,2-disteroyl-3trimethylammonium propane; DOPE, 1,2-sn-dioleoylphoshatidylethanolamine; DC-Chol, 3 P-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol. See, Gao et al., Biochem. Biophys. Res. Comm. 179:280-285 (1991).

As used herein, the term "pharmaceutically acceptable anion" refers to anions of organic and inorganic acids that provide non-toxic salts in pharmaceutical preparations. Examples of such anions include the halides anions, chloride, bromide, and iodide, inorganic anions such as sulfate, phosphate, and nitrate, and organic anions. Organic anions may be derived from simple organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic, acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, and the like. The preparation of pharmaceutically acceptable salts is described in Berge, et al., J. Pharm. Sci. 66:1-19 (1977), incorporated herein by reference.

Physiologically acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low WO 01/93836 PCT/US01/18657 molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA: sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). PEG molecules also contain a fusogenic peptide with an attached Nuclear Localization Signal (NLS) covalently linked to the end of the PEG molecule.

The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DDAB, DMRIE, DODAC, DOGS, DOTAP, DOSPA and DC-Chol. Additionally, a number of commercial preparations of cationic lipids are available that can be used in the present invention. These include, for example, LIPOFECTIN (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, USA); LIPOFECTAMINE (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM (commercially available cationic lipids comprising DOGS in ethanol from Promega Corp., Madison, Wis., USA).

This invention further provides a number of methods for producing micelles with entrapped therapeutic drugs. The method is particularly useful to produce micelles of drugs or compositions having a net overall negative charge, DNA, RNA or negatively charged small molecules. For example, the DNA can be comprised within a plasmid vector and encode for a therapeutic protein, wildtype p53, HSV-tk, p21, Bax, Bad, IL-2, IL-12, GM-CSF, angiostatin, endostatin and oncostatin. In one embodiment, the method requires combining an effective amount of the therapeutic agent with an effective amount of cationic lipids. Cationic lipids useful in the methods of this invention include, but are not limited to, DDAB, dimethyldioctadecyl ammonium bromide; DMRIE: dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane; DOGS: Dioctadecylamidoglycylspermine; DOTAP (same as DOTMA): WO 01/93836 PCT/US01/18657 dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DPTAP: 1,2dipalmitoyl-3-trimethylammonium propane; DSTAP: 1,2-disteroyl-3trimethylammonium propane.

In one aspect, a ratio of from about 30 to about 90% of phosphates contained within the negatively charged therapeutic agent are neutralized by positive charges on lipid molecules (negative charges are in excess) to form an electrostatic micelle complex in an effective concentration of ethanol. In one aspect, the ethanol solution is from about 20% to about 80% ethanol. In a further aspect, the ethanol concentration is about 30%. The ethanol/cationic lipid/therapeutic agent complex is then combined with an effective amount of a fusogenic-karyophilic peptide conjugate. In one aspect, an effective amount of the conjugate is a ratio range from about 0.0 to about 0.3 (positive charges on peptide to negative charges on phosphate groups) to neutralize the majority of the remaining negative charges on the phosphate groups of the therapeutic agents thereby leading to an almost complete neutralization of the complex. The optimal conditions give to the complex a slightly negative charge. However, when the positive charges on cationic lipids exceed the negative charges on the DNA, the excess of positive charges are neutralized by DPPG (dipalmitoyl phosphatidyl glycerol) and its derivatives, or by other anionic lipid molecules in the final micelle complex.

In an alternative embodiment, the above methods can be modified by addition of DNA condensing agents selected from spermine, spermidine, and magnesium or other divalent metal ions neutralizing a certain percentage of phosphate groups.

In a further embodiment, the cationic lipids are combined with an effective amount of fusogenic lipid DOPE at various molar ratios for example, in a molar ratio of from about 1:1 cationic lipid:DOPE. In an alternative embodiment, the cationic lipids are combined with an effective amount of a fusogenic/NLS peptide conjugate. Examples of fusogenic/NLS peptide conjugates include, but are not limited to (KAWLKAF) 3 (SEQ ID NO:1), GLFKAAAKLLKSLWKLLLKA (SEQ ID NO:2), LLLKAFAKLLKSLWKLLLKA (SEQ ID NO:3), as well as all derivatives of the prototype (Hydrophobic3-Karyophilicl-Hydrophobic2- Karyophilicl)2.3 where Hydrophobic is any of the A, I, L, V, P, G, W, F and WO 01/93836 PCT/US01/18657 Karyophilic is any of the K, R, or H, containing a positively-charged residue every 3rd or 4th amino acid, which form alpha helices and direct a net positive charge to the same direction of the helix. Additional examples include but are not limited to GLFKAIAGFIKNGWKGMIDGGGYC (SEQ ID NO:4) from influenza virus hemagglutinin HA-2; YGRKKRRQRRR (SEQ ID NO:5) from TAT of HIV;

MSGTFGGILAGLIGLL(K/R/H)

1 i 6 (SEQ ID NO:6), derived from the N-terminal region of the S protein of duck hepatitis B virus, but with the addition of one to six positively-charged lysine, arginine or histidine residues, and combinations of these, able to interact directly with the phosphate groups of plasmid or oligonucleotide DNA, compensating for part of the positive charges provided by the cationic lipids.

GAAIGLAWIPYFGPAA (SEQ ID NO:7) is derived from the fusogenic peptide of the Ebola virus transmembrane protein; residues 53-70 (C-terminal helix) of apolipoprotein (apo) AII peptide; the 23-residue fusogenic N-terminal peptide of HIV-1 transmembrane glycoprotein gp41; the 29-42-residue fragment from Alzheimer's P-amyloid peptide; the fusion peptide and N-terminal heptad repeat of Sendai virus; the 56-68 helical segment of lecithin cholesterol acyltransferase.

Included within these embodiments are shorter versions of these peptides, that are known to induce fusion of unilamellar lipid vesicles or all that are similarly derivatized with the addition of one to six positively-charged lysine, arginine or histidine residues (K/R/H)1.

6 able to interact directly with the phosphate groups of plasmid or oligonucleotide DNA, compensating for part of the positive charges provided by the cationic lipids. The fusogenic peptides in the fusogenic/NLS conjugates represent hydrophobic amino acid stretches, and smaller fragments of these peptide sequences, that include all signal peptide sequences used in membrane or secreted proteins that insert into the endoplasmic reticulum. Alternatively, the conjugates represent transmembrane domains and smaller fragments of these peptide sequences.

In one aspect of the invention, the NLS peptide component in fusogenic/NLS peptide conjugates is derived from the fusogenic hydrophobic peptides. However, there is an addition of 5-6 amino acid karyophilic Nuclear Localization Signals (NLS) derived from a number of known NLS peptides, as well as from searches of the nuclear protein databases, for stretches of five or more WO 01/93836 PCT/US01/18657 karyophilic amino acid stretches in proteins containing at least four positivelycharged amino aids flanked by a proline or glycine Examples of NLS peptides are shown in Tables 1-8. The NLS peptide component in fusogenic/NLS peptide conjugates are synthetic peptides containing the above said NLS, but further modified by additional K, R, H residues at the central part of the peptide or with P or G at the N- or C-terminus.

In a further aspect, the fusogenic/NLS peptide conjugates are derived from the said fusogenic hydrophobic peptides but with the addition of a stretch of H 4 -6 (four to six histidine residues) in the place of NLS. Micelle formation takes place at pH 5-6 where histidyl residues are positively charged but lose their charge at the nearly neutral pH of the biological fluids, thus releasing the plasmid or oligonucleotide DNA from their electrostatic interaction.

The fusogenic peptide/NLS peptide conjugates are linked to each other with a short amino acid stretch representing an endogenous protease cleavage site.

In a preferred aspect of the invention, the structure of the preferred prototype fusogenic/NLS peptide conjugate used in this invention is: PKKRRGPSP(L/A/I)2- (SEQ ID NO:8), where (L/A/I) 1 2 2 0 is a stretch of 12-20 hydrophobic amino acids containing A, L, I, Y, W, F and other hydrophobic amino acids.

The micelles made by the above methods are further provided by this invention by conversion into liposomes. An effective amount of liposomes (diameter from about 80 to about 160 nm), or of a lipid solution composed of cholesterol (from about 10% to about neutral phospholipid such as hydrogenated soy phosphatidylcholine (HSPC) (from about 40% to about and the derivatized vesicle-forming lipid PEG-DSPE (distearoylphosphatidyl ethanolamine) from about 1-to about 7 mole percent, is added to the micelle solution.

In a specific embodiment, the liposomes are composed of vesicle-forming lipids and between from about 1 to about 7 mole percent of distearoylphosphatidyl ethanolamine (DSPE) derivatized with a polyethyleneglycol. The composition of claim 20, wherein the polyethyleneglycol has a molecular weight is between about 1,000 to 5,000 daltons. Micelles are converted into liposomes with a concomitant decrease of the ethanol concentration which can be accomplished by removal of the WO 01/93836 PCT/US01/18657 ethanol by dialysis of the liposome complexes through permeable membranes or reduced to a diameter of 80-160 nm by extrusion through membranes.

Liposome encapsulated therapeutic agents produced by the above methods are further provided by this invention.

Also provided herein is a method for delivering a therapeutic agent such as plasmid DNA or oligonucleotides to a tissue cell in vivo by intravenous, or other type of injection of the micelles or liposomes. This method specifically targets a primary tumor and the metastases by the long circulating time of the micelle or liposome complex because of the exposure of PEG chains on its surface, its small size (80-160 nm) and the decrease in hydrostatic pressure in the solid tumor from the center to its periphery supporting a preferential extravasation through the tumor vasculature to the extracellular space in tumors. A method for delivering plasmid or oligonucleotide DNA across the cell membrane barrier of the tumors using the micelle or liposome complexes described herein is capable because of the presence of the fusogenic peptides in the complex. In particular, a method for delivering plasmid or oligonucleotide DNA to the liver, spleen and bone marrow after intravenous injection of the complexes is provided. Further provided is a method for delivering therapeutic genes to the liver, spleen and bone marrow of cancer and noncancer patients including but not limited to, factor VIII or IX for the therapy of hemophilias, multidrug resistance, cytokine genes for cancer immunotherapy, genes for the alleviation of pain, genes for the alleviation of diabetes and genes that can be introduced to liver, spleen and bone marrow tissue, to produce a secreted form of a therapeutic protein.

The disclosed therapies also provide methods for reducing tumor size by combining the encapsulated plasmid DNA carrying one or more anticancer genes selected from the group consisting of p53, RB, BRCA1, E1A, bcl-2, MDR-1, p 2 1, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-y, TNF-a, HSV-tk (in combination with ganciclovir), E. coli cytosine deaminase (in combination with 5-fluorocytosine) with encapsulated antisense oligonucleotides (antisense c-fos, c-myc, K-ras), ribozymes or triplex-forming oligonucleotides directed against genes that control the cell cycle or signaling pathways. These methods can be modified by combining the WO 01/93836 PCT/US01/18657 encapsulated plasmid DNA carrying one or more anticancer genes of with encapsulated or free antineoplastic drugs, consisting of the group of adriamycin, angiostatin, azathioprine, bleomycin, busulfane, camptothecin, carboplatin, carmustine, chlorambucile, chlormethamine, chloroquinoxaline sulfonamide, cisplatin, cyclophosphamide, cycloplatam, cytarabine, dacarbazine, dactinomycin, daunorubicin, didox, doxorubicin, endostatin, enloplatin, estramustine, etoposide, extramustinephosphat, flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate, hydroxyurea, idoxuridine, interferons, interleukins, leuprolide, lobaplatin, lomustine, mannomustine, mechlorethamine, mechlorethaminoxide, melphalan, mercaptopurine, methotrexate, mithramycin, mitobronitole, mitomycin, mycophenolic acid, nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine, platinum-triamine complex, plicamycin, prednisolone, prednisone, procarbazine, protein kinase C inhibitors, puromycine, semustine, signal transduction inhibitors, spiroplatin, streptozotocine, stromelysin inhibitors, taxol, tegafur, telomerase inhibitors, teniposide, thalidomide, thiamiprine, thioguanine, thiotepa, tiamiprine, tretamine, triaziquone, trifosfamide, tyrosine kinase inhibitors, uramustine, vidarabine, vinblastine, vinca alcaloids, vincristine, vindesine, vorozole, zeniplatin, zeniplatin, and zinostatin.

The following examples are intended to illustrate, but not limit the invention.

Liposome Composition Liposomes are microscopic vesicles consisting of concentric lipid bilayers.

Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions from a few hundred Angstroms to fractions of a millimeter. Vesicleforming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer. These are neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine phosphatidylinositol and sphingomyelin (SM) and other type of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other WO 01/93836 PCT/US01/18657 lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolaminine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane- 1carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polycthyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles. Preferred lipids for use in the present invention are cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.

Typically, liposomes can be divided into three categories based on their overall size and the nature of the lamellar structure. The three classifications, as developed by the New York Academy Sciences Meeting, "Liposomes and Their Use in Biology and Medicine," December 1977, are multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs).

SUVs range in diameter from approximately 20 to 50 nm and consist of a single lipid bilayer surrounding an aqueous compartment. Unilamellar vesicles can also be prepared in sizes from about 50 nm to 600 nm in diameter. While unilamellar are single compartmental vesicles of fairly uniform size, MLVs vary greatly in size up to 10,000 nm, or thereabouts, are multi-compartmental in their structure and contain more than one bilayer. LUV liposomes are so named because of their large diameter that ranges from about 600 nm to 30,000 nm; they can contain more than one bilayer.

WO 01/93836 PCT/US01/18657 Liposomes may be prepared by a number of methods not all of which produce the three different types of liposomes. For example, ultrasonic dispersion by means of immersing a metal probe directly into a suspension of MLVs is a common way for preparing SUVs.

Preparing liposomes of the MLV class usually involves dissolving the lipids in an appropriate organic solvent and then removing the solvent under a gas or air stream. This leaves behind a thin film of dry lipid on the surface of the container.

An aqueous solution is then introduced into the container with shaking, in order to free lipid material from the sides of the container. This process disperses the lipid, causing it to form into lipid aggregates or liposomes. Liposomes of the LUV variety may be made by slow hydration of a thin layer of lipid with distilled water or an aqueous solution of some sort. Alternatively, liposomes may be prepared by lyophilization. This process comprises drying a solution of lipids to a film under a stream of nitrogen. This film is then dissolved in a volatile solvent, frozen, and placed on a lyophilization apparatus to remove the solvent. To prepare a pharmaceutical formulation containing a drug, a solution of the drug is added to the lyophilized lipids, whereupon liposomes are formed.

Preparing Cationic Liposome/Cationic Peptide/Nucleic Acid Micelles Cationic lipids, with the exception of sphingosine and some lipids in primitive life forms, do not occur in nature. The present invention uses single-chain amphiphiles which are chloride and bromide salts of the alkyltrimethylammonium surfactants including but not limited to C12 and C16 chains abbreviated DDAB (same as DODAB) or CTAB. The molecular geometry of these molecules determines the critical micelle concentration (ratio between free monomers in solution and molecules in micelles). Lipid exchange between the two states is a highly dynamic process; phospholipids have critical micelle concentration values below 10 8 M and are more stable in liposomes; however, single chain detergents, such as stearylamine, may emerge from the liposome membrane upon dilution or intravenous injection in milliseconds (Lasic, 1997).

Cationic lipids include, but are not limited to, DDAB: dimethyldioctadecyl ammonium bromide (same as N,N-distearyl-N,N-dimethylammonium bromide); WO 01/93836 PCT/US01/18657 DMRIE: N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide; DODAC: N,N-dioleyl-N,N-dimethylammonium chloride; DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane; DODAP: 1,2-dioleoyl-3dimethylammonium propane; DOGS: Dioctadecylamidoglycylspermine; DOTAP (same as DOTMA): N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOSPA: N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)- N,N-dimethyl ammonium trifluoroacetate; DPTAP: 1,2- dipalmitoyl-3trimethylammonium propane; DSTAP: 1,2-disteroyl-3-trimethylammonium propane; DC-Chol, 3 -(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol.

Lipid-based vectors used in gene transfer have been formulated in one of two ways. In one method, the nucleic acid is introduced into preformed liposomes made of mixtures of cationic lipids and neutral lipids. The complexes thus formed have undefined and complicated structures and the transfection efficiency is severely reduced by the presence of serum. Preformed liposomes are commercially available as LIPOFECTIN and LIPOFECTAMINE. The second method involves the formation of DNA complexes with mono- or poly-cationic lipids without the presence of a neutral lipid. These complexes are prepared in the presence of ethanol and are not stable in water. Additionally, these complexes are adversely affected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)). An example of a commercially available poly-cationic lipid is TRANSFECTAM. Other efforts to encapsulate DNA in lipid-based formulations have not overcome these problems (see, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer, U.S. Patent No. 4,515,736).

The nucleotide polymers can be single-stranded DNA or RNA, or doublestranded DNA or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and selfreplicating systems such as plasmid DNA. Particularly preferred nucleic acids are plasmids. Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to increase stability, some single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, WO 01/93836 PCT/US01/18657 phosphorodithioate, phosphoroselenate, methylphosphonate, or O-alkyl phosphotriester linkages.

Encapsulating Cationic Liposome/Cationic Peptide/Nucleic Acid Micelles into Neutral Liposomes Cationic lipids used with fusogenic peptide/NLS conjugates to provide the inner layer of the particle can be any of a number of substances selected from the group of DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol. The cationic lipid is combined with DOPE. In one group of embodiments, the preferred cationic lipid is DDAB:DOPE 1:1.

Neutral lipids used herein to provide the outer layer of the particles can be any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids are selected from a group consisting of diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, and cerebrosides. In one group of embodiments, lipids containing saturated, mono-, or di-unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 are preferred. In general, less saturated lipids are more easily sized, particularly when the liposomes must be sized below about 0.16 microns, for purposes of filter sterilization. Consideration of liposome size, rigidity and stability of the liposomes in the final preparation, its shelf life without leakage of the encapsulated DNA, and stability in the bloodstream generally guide the selection of neutral lipids for providing the outer coating of our gene vehicles. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In another group of embodiments, lipids with carbon chain lengths in the range of C14 to C22 are used. Preferably, the neutral lipids used in the present invention are hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and PEGdistearoylphosphatidyl ethanolamine (DSPE) or PEG-ceramide.

Methods for preparing liposomes A variety of methods for preparing various liposome forms have been described in several issued patents, for example, U.S. Patent Nos. 4,229,360; WO 01/93836 PCT/US01/18657 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871; 4,501,728; and 4,837,028, as well as in the articles Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986). These methods do not produce all three different types of liposomes (MLVs, SUVs, LUVs). For example, ultrasonic dispersion by means of immersing a metal probe directly into a suspension of MLVs is a common way for preparing SUVs.

Preparing liposomes of the MLV class usually involves dissolving the lipids in an appropriate organic solvent and then removing the solvent under a gas or air stream. This leaves behind a thin film of dry lipid on the surface of the container.

An aqueous solution is then introduced into the container with shaking, in order to free lipid material from the sides of the container. This process disperses the lipid, causing it to form into lipid aggregates or liposomes. Liposomes of the LUV variety may be made by slow hydration of a thin layer of lipid with distilled water or an aqueous solution of some sort. Alternatively, liposomes may be prepared by lyophilization. This process comprises drying a solution of lipids to a film under a stream of nitrogen. The film is then dissolved in a volatile solvent, frozen, and placed on a lyophilization apparatus to remove the solvent. To prepare a pharmaceutical formulation containing a drug, a solution of the drug is added to the lyophilized lipids, whereupon liposomes are formed.

Following liposome preparation, the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes. Preferably, the preformed liposomes are sized to a mean diameter of about 80 to 160 nm (the upper size limit for filter sterilization before in vivo administration). Several techniques are available for sizing liposomes to a desired size. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 0.05 microns (50 nm) in size. Extrusion of liposome through a small-pore polycarbonate is our preferred method for reducing liposome sizes to a relatively well-defined size distribution.

The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.

One way used to coat DNA with lipid is by controlled detergent depletion from a cationic lipid/DNA/detergent complex. This method can give complexes WO 01/93836 PCT/US01/18657 with stability in plasma. Hofland et al. (1996), have prepared such complexes by dialysis of a mixture of DOSPA/DOPE/DNA/octylglucoside.

Pharmaceutical compositions comprising the cationic liposome/nucleic acid complexes of the invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Generally, normal saline will be employed as the pharmaceutically acceptable carrier.

For in vivo administration, the pharmaceutical compositions are preferably administered parenterally, intravenously, intraperitoneally, subcutaneously, intrathecally, injection to the spinal cord, intramuscularly, intraarticularly, portal vein injection, or intratumorally. More preferably, the pharmaceutical compositions are administered intravenously or intratumorally by a bolus injection. In other methods, the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical "open" or "closed" procedures. The term "topical" means the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, to any surface of the body, nasopharynx, external auditory canal, ocular administration and administration to the surface of any body cavities, inhalation to the lung, genital mucosa and the like.

"Open" procedures are those procedures that include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue.

"Closed" procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via insertion of instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices.

WO 01/93836 PCT/US01/18657

EXAMPLES

Materials and Methods DDAB, DOPE (dioleoylphosphatidylethanolamine) and most other lipids used here were purchased from Avanti Polar Lipids; PEG-DSPE was from Syngena.

Engineering ofplasmid pLF The pGL3-C (Promega) was cut with XbaI and blunt-end ligated using the Klenow fragment of E. coli DNA polymerase. It was then cut with HindIII and the 1689-bp fragment, carrying the luciferase gene, was gel-purified. The pGFP-N1 plasmid (Clontech) was cut with SmaI and HindIII and the 4.7 kb fragment, isolated from an agarose gel, was ligated with the luciferase fragment. JM109 E. coli cells were transformed and 20 colonies were selected; about half of them showed the presence of inserts; 8 clones with inserts were cut with BamHI and XhoI to further confirm the presence of the luciferase gene; seven of them were positive.

Radiolabeled plasmid pLF was generated by culturing Escherichia coli in 3 H-thymidine-5'-triphosphate or 3 2 p inorganic phosphate (5 mCi) (Dupont/NEN, Boston, Mass.) and purified using standard techniques as described above.

DLS measurements A Coulter N4M light scattering instrument was used, at a 900 angle, set at a run time of 200 sec, using 4 to 25 microsec sample time. The scan of the particle size distribution was obtained in 1 ml sample volume using plastic cuvettes, at 20 0

C

and at 0.01 poise viscosity.

In one aspect, this invention provides a method for entrapping DNA into lipids that enhances the content ofplasmid per volume unit, and reduces the toxicity of the cationic lipids used to trap plasmid or oligonucleotide DNA. The DNA becomes hidden in the inner membrane bilayer of the final complex. Furthermore, the gene transfer complex is endowed with long circulation time in body fluids and extravasates preferentially into solid tumors and their metastatic foci and nodules.

The extravasation occurs through their vasculature at most sites of the human or animal body after intravenous injection of the gene-carrying vehicles. This occurs because of their small size (100-160 nm), their content in neutral to slightly WO 01/93836 PCT/US01/18657 negatively-charged lipids in their outer membrane bilayers, and their coating with PEG. These gene delivery vehicles are able to cross the cell membrane barrier after they reach the extracellular tumor space because of the presence of fusogenic peptides conjugated with karyophilic peptides. The vehicles assume a certain predefined orientation in the lipid membrane with their positive ends directed toward DNA and their hydrophobic tail buried inside the hydrophobic lipid bilayer. The labile NLS-fusogenic peptide linkage is cleaved after endocytosis and the remaining NLS peptide bound to plasmid DNA aids its nuclear uptake. This occurs especially when non-dividing cells are targeted, such as liver, spleen or bone marrow cells that represent the major sites for extravasation and concentration of these vehicles other than solid tumors.

Organic solvent A suitable solvent for preparing a micelle from the desired lipid components is ethanol, methanol, or other aliphatic alcohols such as propanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol. Mixtures of two or more solvents may be used in the practice of the invention. It is also to be understood that any solvent that is miscible with an ethanol solution, even in small amounts, can be used to improve micelle formation and its subsequent conversion into liposomes, including chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane, benzene, and toluene.

Cationic lipids In a further embodiment, the liposome encapsulated DNA described herein further comprises an effective amount of cationic lipids. Cationic lipids have been widely used for gene transfer; a number of clinical trials (34 out of 220 total RACapproved protocols as of December, 1997) use cationic lipids. Although many cell culture studies have been documented, systemic delivery of genes with cationic lipids in vivo has been very limited. All clinical protocols use subcutaneous, intradermal, intratumoral, and intracranial injection as well as intranasal, intrapleural, or aerosol administration but not I.V. delivery, because of the toxicity of the cationic lipids and DOPE (see, Martin and Boulikas, 1998). Liposomes WO 01/93836 PCT/US01/18657 formulated from DOPE and cationic lipids based on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-trimethylammonium propane or DOTAP, DMTAP, DPTAP, DSTAP, respectively) or DDAB were highly toxic when incubated in vitro with phagocytic cells (macrophages and U937 cells), but not towards non-phagocytic T lymphocytes. The rank order of toxicity was DOPE/DDAB DOPE/DOTAP DOPE/DMTAP DOPE/DPTAP DOPE/DSTAP; and the toxicity was determined from the effect of the cationic liposomes on the synthesis of nitric oxide (NO) and TNF-a produced by activated macrophages (Filion and Phillips, 1997).

Another aspect to be considered before I.V. injection is undertaken, is that" negatively charged serum proteins can interact and cause inactivation of cationic liposomes (Yang and Huang, 1997). Condensing agents used for plasmid delivery including polylysine, transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE), were found to activate the complement system to varying extents. Strong complement activation was seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS. Modifying the surface of preformed DNA complexes with polyethyleneglycol (Plank et al., 1996) considerably reduced complement activation.

Cationic lipids increase the transfection efficiency by destabilizing the biological membranes, including plasma, endosomal, and lysosomal membranes.

Incubation of isolated lysosomes with low concentrations of DOTAP caused a striking increase in free activity of 1-galactosidase, and even a release of the enzyme into the medium. This demonstrates that the lysosomal membrane is deeply destabilized by the lipid. The mechanism of destabilization was thought to involve an interaction between cationic liposomes and anionic lipids of the lysosomal membrane, thus allowing a fusion between the lipid bilayers. The process was less pronounced at pH 5 than at pH 7.4, and anionic amphipathic lipids were able to prevent partially this membrane destabilization (Wattiaux et al., 1997).

In contrast to DOTAP and DMRIE that were 100% charged at pH 7.4, DC- CHOL was only about 50% charged as monitored by a pH-sensitive fluorophore.

This difference decreases the charge on the external surfaces of the liposomes, and WO 01/93836 PCT/US01/18657 was proposed to promote an easier dissociation ofbilayers containing DC-CHOL from the plasmid DNA, and an increase in release of the DNA-lipid complex into the cytosol from the endosomes (Zuidam and Barenholz, 1997).

Although cationic lipids have been used widely for the delivery of genes, very few studies have used systemic I.V. injection of cationic liposome-plasmid complexes. This is because of the toxicity of the lipid component in animal models, not humans. Administration by I.V. injection of two types of cationic lipids of similar structure, DOTMA and DOTAP, shows that the transfection efficiency is determined mainly by the structure of the cationic lipid and the ratio of cationic lipid to DNA; the luciferase and GFP gene expression in different organs was transient, with a peak level between 4 and 24 hr, dropping to less than 1% of the peak level by day 4 (Song et al., 1997).

A number of different organs in vivo can be targeted after liposomal delivery of genes or oligonucleotides. Intravenous injection of cationic liposome-plasmid complexes by tail vein in mice, targeted mainly the lung and to a smaller extent the liver, spleen, heart, kidney and other organs (Zhu et al., 1993). Intraperitoneal injection of a plasmid-liposome complex expressing antisense K-ras RNA in nude mice inoculated i.p. with AsPC-1 pancreatic cancer cells harboring K-ras point mutations and PCR analysis indicated that the injected DNA was delivered to various organs except brain (Aoki et al., 1995).

A number of factors for DOTAP:cholesterol/DNA complex preparation including the DNA:liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery; maximal gene expression was obtained when a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size were used. Cryo-electron microscopy showed that the DNA was condensed on the interior of invaginated liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (Templeton et al., 1997).

Steps to improve liposome-mediated gene delivery to somatic cells include, persistence of theplasmid in blood circulation, port of entry and transport across the cell membrane, release from endosomal compartments into the cytoplasm, nuclear WO 01/93836 PCT/US01/18657 import by docking through the pore complexes of the nuclear envelope, expression driven by the appropriate promoter/enhancer control elements, and persistence of the plasmid in the nucleus for long periods (Boulikas, 1998a).

Plasmid condensation with spermine In a further embodiment, the liposome encapsulated DNA described herein is condensed with spermine and/or spermidine. DNA can be presented to cells in culture as a complex with polycations such as polylysine, or basic proteins such as protamine, total histones or specific histone fractions, protamine (Boulikas and Martin, 1997). The interaction of plasmid DNA with protamine sulfate, followed by the addition of DOTAP cationic liposomes, offered a better protection of plasmid DNA against enzymatic digestion. The method gave consistently higher gene expression in mice via tail vein injection as compared with DOTAP/DNA complexes. 50 p.g ofluciferase-plasmid per mouse gave 20 ng luciferase protein per mg extracted tissue protein in the lung, that was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. Intraportal injection of protamine/DOTAP/DNA led to about a 100-fold decrease in gene expression in the lung as compared with I.V. injection. Endothelial cells were the primary locus of lacZ transgene expression (Li and Huang, 1997). Protamine sulfate enhanced plasmid delivery into several different types of cells in vitro, using the monovalent cationic liposomal formulations (DC-Chol and lipofectin). This effect was less pronounced with the multivalent cationic liposome formulation, lipofectamine (Sorgi et al., 1997).

Spermine is found to enhance the transfection efficiency ofDNA-cationic liposome complexes in cell culture and in animal studies. This biogenic polyamine at high concentrations caused liposome fusion most likely promoted by the simultaneous interaction of one molecule of spermine (four positively charged amino groups) with the polar head groups of two or more molecules of lipids. At low concentrations (0.03-0.1 mM) it promoted anchorage of the liposome-DNA complex to the surface of cells and enhanced significantly transfection efficiency (Boulikas, unpublished).

WO 01/93836 PCT/US01/18657 The polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the efficiency of adenovirus-mediated gene transfer in cell culture. This was thought to act by neutralizing the negative charges presented by membrane glycoproteins that reduce the efficiency of adenovirus-mediated gene transfer (Arcasoy et al., 1997).

Oligonucleotide transfer In a further embodiment, the liposome encapsulates oligonucleotide DNA.

Encapsulation of oligonucleotides into liposomes increased their therapeutic index, prevented degradation in cultured cells, and in human serum and reduced toxicity to cells (Thierry and Dritschilo, 1992; Capaccioli et al., 1993; Lewis et al., 1996).

However, most studies have been performed in cell culture, and very few in animals in vivo. There are still an important number of improvements needed before these approaches can move into clinical studies.

Zelphati and Szoka (1997), have found that complexes of fluorescently labeled oligonucleotides with DOTAP liposomes, entered the cell using an endocytic pathway mainly involving uncoated vesicles. Oligonucleotides were redistributed from punctate cytoplasmic regions into the nucleus. This process was independent of acidification of the endosomal vesicles. The nuclear uptake of oligonucleotides depended on several factors, such as charge of the particle, where positively charged complexes were required for enhanced nuclear uptake. DOTAP increased over 100 fold the antisense activity of a specific anti-luciferase oligonucleotide.

Physicochemical studies of oligonucleotide-liposome complexes of different cationic lipid compositions indicated that either phosphatidylethanolamine or negative charges on other lipids in the cell membrane are required for efficient fusion with cationic liposome-oligonucleotide complexes to promote entry to the cell (Jaaskelainen et al., 1994).

Similar results were reported by Lappalainen et al. (1997). Digoxigeninlabeled oligodeoxynucleotides (ODNs) complexed with the polycationic DOSPA and the monocationic DDAB (with DOPE as a helper lipid) were taken up by CaSki cells in culture by endocytosis. The nuclear membrane was found to pose a barrier against nuclear import of ODNs that accumulated in the perinuclear area. Although WO 01/93836 PCT/US01/18657 DOSPA/DOPE liposomes could deliver ODNs into the cytosol, they were unable to mediate nuclear import of ODNs. On the contrary, oligonucleotide-DDAB/DOPE complexes with a net positive charge were released from vesicles into the cytoplasm.

It was determined that DDAB/DOPE mediated nuclear import of the oligonucleotides.

DOPE-heme (ferric protoporphyrin IX) conjugates, inserted in cationic lipid particles with DOTAP, protected oligoribonucleotides from degradation in human serum and increased oligoribonucleotide uptake into 2.2.15 human hepatoma cells.

The enhancing effect ofheme was evident only at a net negative charge in the particles (Takle et al., 1997). Uptake of liposomes labeled with "'In and composed of DC-Chol and DOPE was primarily by liver, with some accumulation in spleen and skin and very little in the lung after I.V. tail injection. Preincubation of cationic liposomes with phosphorothioate oligonucleotide induced a dramatic, yet transient, accumulation of the lipid in lung that gradually redistributed to liver. The mechanism of lung uptake involved entrapment of large aggregates of oligonucleotides within pulmonary capillaries at 15 min post-injection via embolism.

Labeled oligonucleotide was localized primarily to phagocytic vacuoles of Kupffer cells at 24 h post-injection. Nuclear uptake of oligonucleotides in vivo was not observed (Litzinger et al., 1996).

Polyethylene glycol (PEG)-coated liposomes In a further embodiment, the liposome encapsulated DNA described herein, further comprise coating of the final complex in step 2 (Fig. 1) with PEG. It is often desirable to conjugate a lipid to a polymer that confers extended half-life, such as polyethylene glycol (PEG). Derivatized lipids that are employed, include PEGmodified DSPE or PEG-ceramide. Addition of PEG components prevents complex aggregation, increases circulation lifetime of particles (liposomes, proteins, other complexes, drugs) and increases the delivery of lipid-nucleic acid complexes to the target tissues. See, Maxfield et al., Polymer 16:505-509 (1975); Bailey, F.E. et al., in: Nonionic Surfactants, Schick, ed., pp. 794-821 (1967); Abuchowski, A. et al., J. Biol. Chem. 252:3582-3586 (1977); Abuchowski, A. et al., Cancer Biochem.

WO 01/93836 PCT/US01/18657 Biophys. 7:175-186 (1984); Katre, N.V. et al., Proc. Natl. Acad. Sci. USA 84:1487- 1491 (1987); Goodson, R. et al. Bio Technology 8:343-346 (1990).

Conjugation to PEG is reported to have reduced immunogenicity and toxicity. See, Abuchowski et al., J. Biol. Chem. 252:3578-3581 (1977). The extent of enhancement of blood circulation time of liposomes, by coating with PEG is described in U.S. Patent No. 5,013,556. Typically, the concentration of the PEGmodified phospholipids, or PEG-ceramide in the complex will be about In a particularly preferred embodiment, the PEG-modified lipid is a PEG-DSPE.

Coating the surface of liposomes with inert materials designed to camouflage the liposome from the body's host defense systems was shown to increase remarkably the plasma longevity of liposomes. The biological paradigm for this "surface modified" sub-branch was the erythrocyte, a cell that is coated with a dense layer of carbohydrate groups, and that manages to evade immune system detection and to circulate for several months (before being removed by the same type of cell responsible for removing liposomes).

The first breakthrough came in 1987 when a glycolipid (the brain tissuederived ganglioside GM1), was identified that, when incorporated within the lipid matrix, allowed liposomes to circulate for many hours in the blood stream (Allen and Chonn, 1987). A second glycolipid, phosphatidylinositol, was also found to impart long plasma residence times to liposomes and, since it was extracted from soybeans, not brain tissue, was believed to be a more pharmaceutically acceptable excipient (Gabizon et al., 1989).

A major advance in the surface-modified sub-branch was the development of polymer-coated liposomes (Allen et al. 1991). Polyethylene glycol (PEG) modification had been used for many years to prolong the half-lives of biological proteins (such as enzymes and growth factors) and to reduce their immunogenicity Beauchamp et al., 1983). It was reported in the early 1990s that PEG-coated liposomes circulated for remarkably long times after intravenous administration.

Half-lives on the order of 24 h were seen in mice and rats, and over 30 hours in dogs.

The term "stealth" was applied to these liposomes because of their ability of evade interception by the immune system. The PEG hydrophilic polymers form dense "conformational clouds" to prevent other macromolecules from interaction with the WO 01/93836 PCT/US01/18657 surface, even at low concentrations of the protecting polymer (Gabizon and Papahadjopoulos, 1988; Papahadjopoulos et al., 1991; reviewed by Torchilin, 1998).

The increased hydrophilicity of the liposomes after their coating with the amphipathic PEG5000 leads to a reduction in nonspecific uptake by the reticuloendothelial system.

Whereas the half-life of antimyosin immunoliposomes was 40 min, by coating with PEG, they increased their half-life to 1000 min after intravenous injection to rabbits (Torchilin et al., 1992).

Micelles, surfactants and small unilamellar vesicles In a further embodiment, the liposome encapsulated DNA described herein, further comprise an initial step of micelle formation between cationic lipids and condensed plasmid or oligonucleotide DNA in ethanol solutions. Micelles are small amphiphilic colloidal particles formed by certain kinds of lipid molecules, detergents or surfactants under defined conditions of concentration, solvent and temperature.

They are composed of a single lipid layer. Micelles can have their hydrophilic head groups assembled exposing their hydrophobic tails to the solvent (for example in aqueous ethanol solution) or can reverse their structures exposing their polar heads toward the solvent such as by lowering the concentration of the ethanol to below 10% (reverse micelles). Micelle systems are in thermodynamic equilibrium with the solvent molecules and environment. This results in constant phase changes, especially upon contact with biological materials, such as upon introduction to cell culture, injection to animals, dilution, contact with proteins or other macromolecules.

These changes result in rapid micelle disassembly or flocculation. This is in contrast to the much higher stability of liposome bilayers.

Single-chain surfactants are able to form micelles (see Table 1, below).

These include the anionic (sodium dodecyl sulfate, cholate or oleate) or cationic (cetyl-trimethylammonium bromide, CTAB) surfactants. CTAB, CTAC, and DOIC micelles yielded larger solubility gaps (lower concentration of colloidally suspended DNA) than corresponding SUV particles containing neutral lipid and CTAB (1:1) (Lasic, 1997).

WO 01/93836 PCT/US01/18657 Table 1: Molecules able to form micelles Molecule Reference CTAB, CTAC, DOIC Lasic, 1997 Detergentiphospholipid micelles Lusa et al.,1998 Dodecyl betaine (amphoteric surfactant) de la Maza et al., 1998 Dodecylphosphocholine cholate Lasic, 1997 Glycine-conjugated bile salt (anionic steroid detergent-like molecule) Leonard and Cohen, 1998 Lipid-dodecyl maltoside micelles Lambert et al., 1998 mixed micelles (Triton X-100 phosphatidylcholine) Lopez et al., 1998 Octylglucoside (non-ionic straight chain detergent) Leonard and Cohen, 1998 Oleate Lasic, 1997 PEG- dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)- Tirosh et al., 1998 PEG2000) Phosphatidylcholine (neutral zwitterionic) Schroeder et al., 1990 Polyethyleneglycol (MW 5000)-distearoyl phosphatidyl ethanolamine Weissig et al., 1998

(PEG-DSPE)

sodium dodecyl sulfate (anionic straight chain detergent) Leonard and Cohen, 1998 Sodium taurofusidate (conjugated fungal bile salt analog) Leonard and Cohen, 1998 Taurine- conjugated bile salts (anionic steroid detergent-like Leonard and Cohen, 1998 molecule) Triton X-100 surfactant Lasic, 1997 There is a critical detergent/phospholipid ratio at which lamellar-to-micellar transition occurs. For example, the vesicle-micelle transition was observed for dodecyl maltoside with large unilamellar liposomes. A striking feature of the solubilization process by dodecyl maltoside was the discovery of a new phase, consisting of a very viscous "gel-like" structure composed of long filamentous thread-like micelles, over 1 to 2 microns in length.

A long circulating complex needs to be slightly anionic. Therefore the liposomes used for the conversion of the micelles into liposomes contain bipolar lipids (PC, PE) and 1-30% negatively charged lipids (DPPG). The cationic lipids which are toxic, are hidden in the inner liposome membrane bilayer. Those reaching the solid tumor will exert their toxic effects causing apoptosis. Apoptosis will be caused by the delivery of the toxic drug or anti-neoplastic gene or oligonucleotide to the cancer cell but also by the nuclear localization of the cationic lipids (along with plasmid DNA) to the nucleus. Indeed, a number of studies suggest that plasmid DNA is imported to nuclei; its translocation docks cationic lipid molecules electrostatically attached to the DNA. These cationic lipid molecules exert their toxicity by interfering with the nucleosome and domain structure of the chromatin causing local destabilization. This disturbance or aberrant chromatin reorganization WO 01/93836 PCT/US01/18657 could be exerted at the level of the nuclear matrix where plasmid DNA is attached for transcription, autonomous replication, or integration via recombination.

Surfactants have found wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in: Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, 1988, p. 285).

Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general, their HLB values range from 2 to about 18, depending on their structure. Nonionic surfactants include, nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated, block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

Classical micelles may not be effective as gene transfer vehicles, but important intermediates in the formation of liposome complexes encapsulating drugs or nucleic acids. The stability of single chain surfactants-DNA-colloidal systems is lower than SUV particles containing neutral lipid and CTAB However, WO 01/93836 PCT/US01/18657 second generation micelles are able to target tumors in vivo. Weissig and coworkers (1998) used the soybean trypsin inhibitor (STI) as a model protein to target tumors. STI was modified with a hydrophobic residue of N-glutaryl-phosphatidylethanolamine (NGPE) and incorporated into both polyethyleneglycol (MW 5000)distearoyl phosphatidyl ethanolamine (PEG-DSPE) micelles 20 nm) and PEG- DSPE-modified long-circulating liposomes (ca. 100 nm). As determined from the protein label by using 1 'In attached to soybean trypsin inhibitor via protein-attached diethylene triamine pentaacetic acid, DTPA, PEG-lipid micelles accumulated better than the same protein anchored in long-circulating PEG-liposomes in subcutaneously established Lewis lung carcinoma in mice after tail vein injection.

Loading a liposomal dispersion with an amphiphilic drug may cause a phase transformation into a micellar solution. The transition from high ratios of phospholipid to drug (from 2:1 to 1:1 downwards) were accompanied by the conversion of liposomal dispersions of milky-white appearance (particle size 200 nm) to nearly transparent micelles (particle size below 25 nm). See, Schutze and Muller-Goymann (1998).

Fusogenic peptides In a further embodiment, the liposome encapsulated DNA described herein further comprises an effective amount of a fusogenic peptide. Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. This hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion (Decout et al., 1999).

Hemagglutinin (HA) is a homotrimeric surface glycoprotein of the influenza virus. In infection, it induces membrane fusion between viral and endosomal membranes at low pH. Each monomer consists of the receptor-binding HA1 domain and the membrane-interacting HA2 domain. The NH 2 -terminal region of the HA2 domain (amino acids 1 to 127), the so-called "fusion peptide," inserts into the target membrane and plays a crucial role in triggering fusion between the viral and endosomal membranes. Based on the substitution of eight amino acids in region 14 with cysteines and spin-labeling electron paramagnetic resonance, it was WO 01/93836 PCT/US01/18657 concluded that the peptide forms an alpha-helix tilted approximately 25 degrees from the horizontal plane of the membrane with a maximum depth of 15 A from the phosphate group (Macosko et al., 1997). Use of fusogenic peptides from influenza virus hemagglutinin HA-2 enhanced greatly the efficiency of transferrin-polylysine- DNA complex uptake by cells. The peptide was linked to polylysine and the complex was delivered by the transferrin receptor-mediated endocytosis (reviewed by Boulikas, 1998a). This peptide has the sequence: GLFEAIAGFI ENGWEGMIDG GGYC (SEQ ID NO:9) and is able to induce the release of the fluorescent dye calcein from liposomes prepared with egg yolk phosphatidylcholine, which was higher at acidic pH. This peptide was also able to increase up to the anti-HIV potency of antisense oligonucleotides, at a concentration of 0.1-1 mM, using CEM-SS lymphocytes in culture. This peptide changes conformation at the slightly more acidic environment of the endosome, destabilizing and breaking the endosomal membrane (reviewed by Boulikas, 1998a).

The presence of negatively charged lipids in the membrane is important for the manifestation of the fusogenic properties of some peptides, but not of others.

Whereas the fusogenic action of a peptide, representing a putative fusion domain of fertilin, a sperm surface protein involved in sperm-egg fusion, was dependent upon the presence of negatively charged lipids, that of the HIV2 peptide was not (Martin and Ruysschaert, 1997).

For example, to analyze the two domains on the fusogenic peptides of influenza virus hemagglutinin HA, HA-chimeras were designed in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. Constructs of HA were made in which the cytoplasmic tail was replaced by peptides of human neurofibromin type 1 (NFI) (residues 1441 to 1518) or c-Raf-1, (residues 51 to 131) and were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins showed that, after the pH was lowered, a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-I cells took place.

WO 01/93836 PCT/US01/18657 This indicated that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore (Schroth-Diaz et al., 1998).

A remarkable discovery was that the TAT protein of HIV is able to cross cell membranes (Green and Loewenstein, 1998) and that a 36-amino acid domain of TAT, when chemically cross-linked to heterologous proteins, conferred the ability to transduce into cells. The 11-amino acid fusogenic peptide of TAT (YGRKKRRQRRR (SEQ ID NO:10)) is a nucleolar localization signal (see Boulikas, 1998b).

Another protein of HIV, the glycoprotein gp41, contains fusogenic peptides.

Linear peptides derived from the membrane proximal region of the gp41 ectodomain have potential applications as anti-HIV agents and inhibit infectivity by adopting a helical conformation (Judice et al., 1997). The 23 amino acid residue, N-terminal peptide of HIV-1 gp41 has the capacity to destabilize negatively charged large unilamellar vesicles. In the absence of cations, the main structure was a poreforming alpha-helix, whereas in the presence of Ca2+ the conformation switched to a fusogenic, predominantly extended beta-type structure. The fusion activity of HIV(ala) (bearing the R22-A substitution) was reduced by 70%, whereas fusogenicity was completely abolished when a second substitution was included, arguing that it is not an alpha-helical but an extended structure adopted by the HIV-1 fusion peptide that actively destabilizes cholesterol-containing, electrically neutral membranes (Pereira et al., 1997).

The prion protein (PrP) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in humans, where PrP is converted into an altered form (termed PrPSc). According to computer modeling calculations, the 120 to 133 and 118 to 135 domains of PrP are tilted lipidassociating peptides inserting in a oblique way into a lipid bilayer and able to interact with liposomes to induce leakage of encapsulated calcein (Pillot et al., 1997b).

The C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29and 29-42) have properties related to those of the fusion peptides of viral proteins inducing fusion of liposomes in vitro. These properties could mediate a direct WO 01/93836 PCT/US01/18657 interaction of the amyloid peptide with cell membranes and account for part of the cytotoxicity of the amyloid peptide. In view of the epidemiologic and biochemical linkages between the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, examination of the potential interaction between the three common apoE isoforms and the C-terminal fragments of the amyloid peptide showed that only apoE2 and apoE3, not apoE4, are potent inhibitors of the amyloid peptide fusogenic and aggregational properties. The protective effect of apoE against the formation of amyloid aggregates was thought to be mediated by the formation of stable apoE/amyloid peptide complexes (Pillot et al., 1997a; Lins et al., 1999).

The fusogenic properties of an amphipathic net-negative peptide (WAE 11), consisting of 11 amino acid residues were strongly promoted when the peptide was anchored to a liposomal membrane. The fusion activity of the peptide appeared to be independent ofpH and membrane merging, and the target membranes required a positive charge that was provided by incorporating lysine-coupled phosphatidylethanolamine Whereas the coupled peptide could cause vesicle aggregation via nonspecific electrostatic interaction with PE-K, the free peptide failed to induce aggregation of PE-K vesicles (Pecheur et al., 1997).

A number of studies suggest that stabilization of an alpha-helical secondary structure of the peptide after insertion in lipid bilayers in membranes of cells or liposomes is responsible for the membrane fusion properties ofpeptides. Zn2+ enhances the fusogenic activity ofpeptides because it stabilizes the alpha-helical structure. For example, the HEXXH (SEQ ID NO: 11) domain of the salivary antimicrobial peptide, located in the C-terminal functional domain ofhistatin-5, a recognized zinc-binding motif is in a helicoidal conformation (Martin et al., 1999; Melino et al., 1999; Curtain et al., 1999).

Fusion peptides have been formulated with DNA plasmids to create peptidebased gene delivery systems. A combination of the YKAKnWK (SEQ ID NO: 12) peptide, used to condense plasmids into 40 to 200 nm nanoparticles, with the GLFEALLELLESLWELLLEA (SEQ ID NO:13) amphipathic peptide, that is a pHsensitive lytic agent designed to facilitate release of the plasmid from endosomes enhanced expression systems containing the beta-galactosidase reporter gene (Duguid et al., 1998). See Table 2, below.

WO 01/93836 PCT/US01/18657 Table 2. Fusogenic peptides Fusogenic peptide Source Protein Properties Reference GLFEAIAGFIENGWEG Influenza virus Endowed with membrane Bongartz et al., 1994 MIDGGGYC (SEQ ID hemagglutinin fusion properties NO:9) HA-2 YGRKKRRQRRR (SEQ TAT of HIV Endowed with membrane Green and ID NO:5) fusion properties Loewenstein, 1988 23-residue fusogenic N- HIV-1 trans- Was able to insert as an Curtain et al., 1999 terminal peptide membrane alpha-helix into neutral glycoprotein gp41 phospholipid bilayers residue peptide (SV- Fusion peptide Induced lipid mixing of egg Ghosh and Shai, 117) and N-terminal phosphatidylcholine- 1999 heptad repeat of phosphatidyiglycerol Sendai virus (PC/PG) large unilamellar vesicles (LUVs) 23 hydrophobic amino S protein of A high degree of similarity Rodriguez-Crespo et acids in the amino-terminal hepatitis B virus with known fusogenic al., 1994 region (HBV) peptides from other viruses.

MSGTFGGILAGLIGLL N-terminal region Was inserted into the Rodriguez-Crespo et (SEQ ID NO:6) of the S protein of hydrophobic core of the al., 1999 duck hepatitis B lipid bilayer and induced Virus (DHBV) leakage of internal aqueous contents from both neutral and negatively charged liposomes MSPSSLLGLLAGLQV S protein of Was inserted into the Rodriguez-Crespo et, (SEQ ID NO: 14) woodchuck hydrophobic core of the al., 1999 hepatitis B virus lipid bilayer and induced (WHV) leakage of internal aqueous contents from both neutral and negatively charged liposomes N-terminus of Nef Nef protein of Membrane-perturbing and Macreadie et al., human fusogenic activities in 1997 immunodeficienc artificial membranes; causes y type 1 (HIV-1) cell killing in E. coli and yeast Amino-terminal sequence Fl polypeptide of Can be used as a carrier Partidos et al., 1996 F1 polypeptide measles virus system for CTL epitopes

(MV)

19-27 amino acid segment Glycoprotein Adopts an amphiphilic Voneche et al., 1992 gp51 of bovine structure and plays a key leukemia virus role in the fusion events induced by bovine leukemia virus 120 to 133 and 118 to 135 Prion protein Tilted lipid-associating Pillot et al., 1997b domains peptide; interact with liposomes to induce leakage of encapsulated calcein 29-42-residue fragment Alzheimer's beta- Endowed with capacities Lins et al., 1999 amyloid peptide resembling those of the tilted fragment of viral fusion proteins Non-aggregated amyloid Alzheimer's beta- Induces apoptotic neuronal Pillot et al., 1999 beta-peptide (1-40) amyloid peptide cell death____ WO 01/93836 PCT/US01/18657 Fusogenic peptide Source Protein Properties Reference LCAT 56-68 helical Lecithin Forms stable beta-sheets in Peelman et al., 1999; segment cholesterol lipids Decout et al., 1999 acyltransferase

(LCAT)

Peptide sequence B 18 Membrane- Triggers fusion between Ulrich et al., 1999 associated sea lipid vesicles; a histidineurchin sperm rich motif for binding zinc protein binding is required for the fusogenic function 53-70 (C-terminal helix) Apolipoprotein Induces fusion of Lambert et al., 1998 (apo) All unilamellar lipid vesicles and displaces apo Al from HDL and r-HDL Residues 90-111 PH-30 alpha (a Membrane-fusogenic Niidome et al., 1997 protein activity to acidic functioning in phospholipid bilayers sperm-egg fusion) Casein signal peptides Alpha s2- and Interact with Creuzenet et al., 1997 beta-casein dimyristoylphosphatidylglycerol and -choline liposomes; show both lytic and fusogenic activities Pardaxin Amphipathic Forms voltage-gated, Lelkes and polypeptide, cation-selective pores; Lazarovici, 1988 purified from the mediated the aggregation of gland secretion of liposomes composed of the Red Sea phosphatidylserine but not Moses sole of phosphatidylcholine flatfish Pardachirus marmoratus Salivary Aggregates and fuses Melino et al., 1999 antimicrobial negatively charged small peptide unilamellar vesicles in the presence of Zn2+ Gramicidin (linear Antibiotic Induces aggregation and Massari and Colonna, hydrophobic polypeptide) fusion of vesicles 1986; Toumois et al., 1990 Amphipathic negatively Synthetic Forms an alpha-helix Martin et al., 1999 charged peptide consisting inserted and anchored into of 11 residues (WAE) the membrane (favored at 37oC) oriented almost parallel to the lipid acyl chains; promotes fusion of large unilamellar liposomes

(LUV)

A polymer of polylysine Synthetic Histidyl residues become Midoux and (average 190) partially cationic upon protonation of Monsigny, 1999 substituted with histidyl the imidazole groups at pH residues below disrupt endosomal membranes GLFEALLELLESLWELL Synthetic Amphipathic peptide; a pH- Duguid et al., 1998 LEA (SEQ ID NO:4) sensitive lytic agent to facilitate release of the plasmid from endosomes WO 01/93836 PCT/US01/18657 Fusogenic peptide Source Protein Properties Reference

(LKKL)

4 (SEQ ID NO:15) Synthetic Amphiphilic fusogenic Gupta and Kothekar, peptide, able to interact with 1997 four molecules of DMPC Ac-(Leu-Ala-Arg-Leu) 3 Synthetic; basic Caused a leakage of Suenaga et al., 1989;

NHCH

3 (SEQ ID NO:16) amphipathic contents from small Lee et al., 1992 peptides unilamellar vesicles composed of egg yolk phosphatidylcholine and egg yolk phosphatidic acid (3:1) Amphiphilic anionic Synthetic Can mimic the fusogenic Murata et al., 1991 peptides E5 and E5L activity of influenza hemagglutinin (HA) acid peptide with Synthetic; Becomes an amphipathic Parente et al., 1988 the major repeat unit Glu- designed to mimic alpha-helix as the pH is Ala-Leu-Ala (GALA) 7 the behavior of lowered to 5.0 fusion of (SEQ ID NO: 17) the fusogenic phosphatidylcholine small sequences of viral unilamellar vesicles induced fusion proteins by GALA requires a peptide length greater than 16 amino acids Poly Glu-Aib-Leu-Aib Synthetic Amphiphilic structure upon Kono et al., 1993 (SEQ ID NO: 18) Aib the formation of alpharepresents 2- helix; caused fusion of aminoisobutyric acid EYPC liposomes and dipalmitoylphosphatidylchol ine liposomes more strongly with decreasing pH Fusogenic lipids DOPE is a fusogenic lipid; elastase cleavage of N-methoxy-succinyl-Ala- Ala-Pro-Val-DOPE (SEQ ID NO:19) converted this derivative to DOPE (overall positive charge) to deliver an encapsulated fluorescent probe, calcein, into the cell cytoplasm (Pak et al., 1999). An oligodeoxynucleic sequence of 30 bases complementary to a region ofbeta-endorphin mRNA elicited a concentrationdependent inhibition ofbeta-endorphin production in cell culture after it was encapsulated within small unilamellar vesicles (50 nm) containing dipalmitoyl-DLalpha-phosphatidyl-L-serine endowed with fusogenic properties (Fresta et al., 1998).

Nuclear localization signals (NLS) In a further embodiment, the liposome encapsulated plasmid or oligonucleotide DNA described herein further comprise an effective amount of nuclear localization signal (NLS) peptides. Trafficking of nuclear proteins from the site of their synthesis in the cytoplasm to the sites of function in the nucleus through WO 01/93836 PCT/US01/18657 pore complexes is mediated by NLSs on proteins to be imported into nuclei (Tables 3-10, below). Protein translocation from the cytoplasm to the nucleoplasm involves: the formation of a complex of karyopherin a with NLS-protein; (ii) subsequent binding of karyopherin p; (iii) binding of the complex to FXFG peptide repeats on nucleoporins; (iv) docking of Ran-GDP to nucleoporin and to karyopherin heterodimer by p10; a number of association-dissociation reactions on nucleoporins that dock the import substrate toward the nucleoplasmic side with a concomitant GDP-GTP exchange reaction transforming Ran-GDP into Ran-GTP and catalyzed by karyopherin a; and (vi) dissociation from karyopherin p and release of the karyopherin a/NLS-protein by Ran-GTP to the nucleoplasm.

Karyophilic and acidic clusters were found in most non-membrane serine/threonine protein kinases whose primary structure has been examined (Table These karyophilic clusters might mediate the anchoring of the kinase molecules to transporter proteins for their regulated nuclear import and might constitute the nuclear localization signals. In contrast to protein transcription factors that are exclusively nuclear possessing strong karyophilic peptides composed of at least four arginines, and lysines, within an hexapeptide flanked by proline and glycine helix-breakers, protein kinases often contain one histidine and three K+R residues (Boulikas, 1996). This was proposed to specify a weak NLS structure resulting in the nuclear import of a fraction of the total cytoplasmic kinase molecules, as well as in their weak retention in the different ionic strength nuclear environment. Putative NLS peptides in protein kinases may also contain hydrophobic or bulky aromatic amino acids proposed to further diminish their capacity to act as strong NLS.

Most mammalian proteins that participate in DNA repair pathways seem to possess strong karyophilic clusters containing at least four R+K over a stretch of six amino acids (Table 7).

Rules to predict nuclear localization of an unknown protein Several simple rules have been proposed for the prediction of the nuclear localization of a protein of an unknown function from its amino acid sequence: WO 01/93836 PCT/US01/18657 An NLS is defined as four arginines plus lysines within an hexapeptide; the presence of one or more histidines in the tetrad of the karyophilic hexapeptide, often found in protein kinases that have a cytoplasmic and a nuclear function, may specify a weak NLS whose function might be regulated by phosphorylation or may specify proteins that function in both the cytoplasm and the nucleus (Boulikas, 1996); (ii) The K/R clusters are flanked by the a-helix breakers G and P thus placing the NLS at a helix-turn-helix or end of a a-helix. Negatively-charged amino acids E) are often found at the flank of the NLS and on some occasions may interrupt the positively-charged NLS cluster; (iii) Bulky amino acids F, Y) are not present within the NLS hexapeptide; (iv) NLS signals may not be flanked by long stretches ofhydrophobic amino acids five); a mixture of charged and hydrophobic amino acids serves as a mitochondrial targeting signal; The higher the number of NLSs, the more readily a molecule is imported to the nucleus (Dworetzky et al., 1988). Even small proteins, for example histones (10-22 kDa), need to be actively imported to increase their import rates compared with the slow rate of diffusion of small molecules through pores; (vi) Signal peptides are stronger determinants than NLSs for protein trafficking. Signal peptides direct proteins to the lumen of the endoplasmic reticulum for their secretion or insertion into cellular membranes (presence of transmembrane domains) (Boulikas, 1994); (vii) Signals for the mitochondrial import of proteins (a mixture of hydrophobic and karyophilic amino acids) may antagonize nuclear import signals and proteins possessing both type of signals may be translocated to both mitochondria and nuclei; (viii) Strong association of a protein with large cytoplasmic structures (membrane proteins, intermediate filaments) make such proteins unavailable for import even though they posses NLS-like peptides (Boulikas, 1994); Transcription factors and other nuclear proteins posses a great different number of putative NLS stretches. Of the sixteen possible forms of putative NLS WO 01/93836 PCT/US01/18657 structures the most abundant types are the 0096x, 6000x, 0000, and 66x6x9, where 6 is R or K, together accounting for about 70% of all karyophilic clusters on transcription factors (Boulikas, 1994); A small number of nuclear proteins seem to be void of a typical karyophilic NLS. Either non karyophilic peptides function for their nuclear import, as such molecules possess bipartite NLSs, or these NLS-less proteins depend absolutely for import on their strong complexation in the cytoplasm with a nuclear protein partner able to be imported (Boulikas, 1994). This mechanism may ensure a certain stoichiometric ratio of the two molecules in the nucleus, and might be of physiological significance; and (xi) A number of proteins may be imported via other mechanisms not dependent on classical NLS.

A number of processes have been found to be regulated by nuclear import including nuclear translocation of the transcription factors NF-KB, rNFIL-6, ISGF3, SRF, c-Fos, GR as well as human cyclins A and B1, casein kinase II, cAMPdependent protein kinase II, protein kinase C, ERK1 and ERK2. Failure of cells to import specific proteins into nuclei can lead to carcinogenesis. For example, BRCA1 is mainly localized in the cytoplasm in breast and ovarian cancer cells, whereas in normal cells the protein is nuclear. mRNA is exported through the same route as a complex with nuclear proteins possessing nuclear export signals (NES).

The majority of proteins with NES are RNA-binding proteins that bind to and escort RNAs to the cytoplasm. However, other proteins with NES function in the export of proteins; CRM1, that binds to the NES sequence on other proteins and interacts with the nuclear pore complex, is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells. Nuclear localization and export signals (NLS and NES) are found on a number of important molecules, including p 53 v-Rel, the transcription factor NF-ATc, the c-Abl nonreceptor tyrosine kinase, and the fragile X syndrome mental retardation gene product. The deregulation of their normal import/export trafficking has important implications for human disease. Both nuclear import and export processes can be manipulated by conjugation of proteins with NLS or NES peptides. During gene therapy, the foreign DNA needs to enter nuclei for its transcription. A pathway is proposed involving the complexation of WO 01/93836 PCT/US01/18657 plasmids and oligonucleotides with nascent nuclear proteins possessing NLSs as a prerequisite for their nuclear import. Covalent linkage ofNLS peptides to oligonucleotides and plasmids or formation of complexes of plasmids with proteins possessing multiple NLS peptides was proposed (Boulikas, 1998b) to increase their import rates and the efficiency of gene expression. Cancer cells were predicted to import more efficiently foreign DNA into nuclei, compared with terminally differentiated cells because of their increased rates of proliferation and protein import.

Antineoplastic drugs In a further embodiment, the liposome encapsulated plasmid or oligonucleotide DNA described herein, further comprises its use for reducing tumor size or restricting its growth with combination with encapsulated or free antineoplastic agents. Antineoplastic agents preferably are: alkylating agents having the bis-(2-chloroethyl)-amine group such as chlormethine, chlorambucile, melphalan, uramustine, mannomustine, extramustinephosphat, mechlorethaminoxide, cyclophosphamide, ifosfamide, or trifosfamide; (ii) alkylating agents having a substituted aziridine group, for example tretamine, thiotepa, triaziquone, or mitomycine; (iii) alkylating agents of the methanesulfonic ester type such as busilfane; (iv) alkylating N-alkyl-N-nitrosourea derivatives, for example carmustine, lomustine, semustine, or streptozotocine; alkylating agents of the mitobronitole, dacarbazine, or procarbazine type; (vi) complexing agents such as cisplatin; (vii) antimetabolites of the folic acid type, for example methotrexate; (viii) purine derivatives such as mercaptopurine, thioguanine, azathioprine, tiamiprine, vidarabine, or puromycine and purine nucleoside phosphorylase inhibitors; (ix) pyrimidine derivatives, for example fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine, flucytosine; antibiotics such as dactinomycin, daunorubicin, doxorubicin, mithramycin, bleomycin or etoposide; (xi) vinca alkaloids; (xii) inhibitors of proteins overexpressed in cancer cells such as telomerase inhibitors, glutathione inhibitors, proteasome inhibitors; (xiii) modulators or inhibitors of signal transduction pathways such as phosphatase inhibitors, protein kinase C inhibitors, casein kinase inhibitors, insulin-like growth factor-1 receptor inhibitor, ras WO 01/93836 PCT/US01/18657 inhibitors, ras-GAP inhibitor, protein tyrosine phosphatase inhibitors; (xiv) tumor angiogenesis inhibitors such as angiostatin, oncostatin, endostatin, thalidomide; (xv) modulators of the immune response and cytokines such as interferons, interleukins, TNF-alpha; (xvi) modulators of the extracellular matrix such as matrix metalloproteinase inhibitors, stromelysin inhibitors, plasminogen activator inhibitor; (xvii) hormone modulators for hormone-dependent cancers (breast cancer, prostate cancer) such as antiandrogen, estrogens; (xviii) apoptosis regulators; (xix) bFGF inhibitor; (xx) multiple drug resistance gene inhibitor; (xxi) monoclonal antibodies or antibody fragments against antigenes overexpressed in cancer cells (anti-Her2/neu for breast cancer); (xxii) anticancer genes whose expression will cause apoptosis, arrest the cell cycle, induce an immune response against cancer cells, inhibit tumor angiogenesis i.e. formation of blood vessels, tumor suppressor genes (p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-y, and TNF-a); and (xxiii) antisense oligonucleotides (antisense c-fos, c-myc, K-ras). Optionally these drugs are administered in combination with chlormethamine, prednisolone, prednisone, or procarbazine or combined with radiation therapy. Future new anticancer drugs added to the arsenal are expected to be ribozymes, triplex-forming oligonucleotides, gene inactivating oligonucleotides, a number of new genes directed against genes that control the cell proliferation or signaling pathways, and compounds that block signal transduction.

Anti-cancer drugs include: acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, adriamycin, aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, WO 01/93836 WO 0193836PCT/US01/18657 droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemeitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interferon alfa-2a, interferon a-2b, interferon a-ni1, interferon a-n' interferon P-1 a, interferon y-i b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, meiphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomnestane, porfimer sodium, porfiromycin, prednimustine, prednisone, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, safingol, safingol hydrochloride, semustine, simntrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin, taxol, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan hydrochloride, toremifene citrate, trestolone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinbiastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride.

Other anti-cancer drugs include: 20-epi- 1,25 dihydroxyvitamin D3, ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adozelesin, WO 01/93836 WO 0193836PCT/USOI/18657 aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, arnrubicin, amnsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-i, antiandrogen, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCRIABL antagonists, benzochlorins, benzoylstaurosporine, beta lactamn derivatives, betaalethine, betaclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, caiphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinase inhibitors (ICOS), castanospermine, cecropin B, cetrorelix, chlorlns, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomifene analogues, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, cramrbescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemnycin, cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydrotaxol, 9-dioxamycin, diphenyl spiromustine, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epristeride, estramustine analogue, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemnestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadoliniumn gallium nitrate texaphyrin, galocitabine, ganirelix, gelatinase inhibitors, gemcitabinc, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, WO 01/93836 PCT/USO1/18657 imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor- 1 receptorinhibitor, interferon agonists, interferons, interleukins, lobenguane, lododoxorubicin, ipomeanol, irinotecan, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, j asplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptoistatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarnzole, linear polyamine analogue, lipophilic disaccharide peptide, lipophulic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marirastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide, MIE inhibitor, mifepristone, niltefosine, mirimostim, mismatched double stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, myriaporone, Nacetyldinaline, N-substituted benzamides, nafarelin, nagrestip, naloxone +pentazocine, napavin, naphterpin, nartograstir, nedaplatin, nemonibicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, 06-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel analogues, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin, pentrozole, perfiubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinumtriamine complex, porfimer sodium, porfiromycin, propyl bis-acridone, WO 01/93836 PCT/US01/18657 prostaglandin J2, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal., protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin, ribozymes, RII retinamide, rogletimide, rohitukine, romurtide, roquinimex, rubiginone B 1, ruboxyl, safingol, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, velaresol, veramine, verdins, verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin stimalamer.

pH-sensitive peptide-DNA complexes In a further embodiment of the invention, the genes in plasmid DNA are brought in interaction with fusogenic peptide/NLS conjugates. In a further embodiment the NLS moiety is a stretch of histidyl residues able to assume a net WO 01/93836 PCT/US01/18657 positive charge at a pH of about 5 to 6 and to show a reduction or loose completely this charge at pH above 7. The electrostatic interaction of these positively-charged peptides with the negatively-charged plasmid DNA molecules, established at pH 5-6 is weakened at physiological pH (pH-sensitive peptide-DNA complexes).

The first step of the present invention involves complex formation between the plasmid or oligonucleotide DNA with the histidyl/fusogenic peptide conjugate and lipid components in 10-90% ethanol at pH 5.0 to 6.0. The conditions must be where the histidyl residues have a net positive charge and can establish electrostatic interactions with plasmids, oligonucleotides or negatively-charged drugs. At the same time, the presence of the positively-charged lipid molecules promotes formation of micelles. At the second step, micelles are converted into liposomes by dilution with water and mixing with pre-made liposomes or lipids at pH 5-6. This is followed by dialysis against pH 7 and extrusion through membranes, entrapping and encapsulating plasmids or oligonucleotides to with a very high yield.

Whereas the composition ofpeptides and cationic lipids in the first step provides the lipids of the internal bilayer, the type of liposomes or lipids added at step 2 provide the external coating of the final liposome formulation (Figure 1).

Examples for the formulations of peptides include: HHHHHSPSL16(SEQ ID NO:623), and HHHHHSPS(LAI) 5 (SEQ ID NO:624).

These are added at a 1:0.5:0.5 molar ratio (negative charge on DNA: cationic liposome: histidine peptide). The peptide inserts in an alpha-helical conformation inside the lipid bilayer and not only carries out DNA condensation but also endows membrane fusion properties to the complex to improve entrance across the cell membrane. The type of hydrophobic amino acids (for example, content in aromatic amino acids), in the peptide chain is very important as is the length of the peptide chain in ensuring integrity and rigidity of the complexes. Coating the outer surface of the complexes with polyethyleneglycol, hyaluronic acids and other polymers conjugated to lipids gives the particles long circulation properties in body fluids and the ability to target solid tumors and their metastases after intravenous injection, and also the ability to cross the tumor cell membrane.

WO 01/93836 PCT/US01/18657 Protease-sensitive linkages in peptides between the NLS and fusogenic moieties Conversion of micelles into liposomes An important issue of the present invention is the conversion of micelles formed between the DNA and the cationic lipids, in the presence of ethanol, into liposomes. This is done by the direct addition of the micelle complex into an aqueous solution of preformed liposomes. The liposomes have an average size of 80-160 nm or vice versa, leading to a solution of a final ethanol concentration below A formulation suitable for pharmaceutical use and for injection into humans and animals will require that the liposomes are of neutral composition (such as cholesterol, PE, PC) coated with PEG.

However, another important aspect is the research application of the present invention, such as for transfection of cells in culture. The composition of the aqueous solution of liposomes is any type of liposomes containing cationic lipids and suitable therefore for transfection of cells in culture such as DDAB:DOPE 1:1.

These liposomes are pre-formed and downsized by sonication or extrusion through membranes to a diameter of 80-160 nm. The ethanolic micelle preparations are then added to the aqueous solution of liposomes with a concomitant dilution of the ethanol solution to below 10%. This step will result in further condensation of DNA or interaction of the negatively-charged phosphate groups on DNA with positively charged groups on lipids. Care must be taken so as only part of the negative charges on DNA are neutralized by lipids in the micelle. The remaining charge neutralization of the DNA is to be provided by the cationic component of the preformed liposomes in the second step.

Regulatory DNA and nuclear matrix-attached DNA In a further embodiment of the present invention, the genes in plasmid DNA are driven by regulatory DNA sequences isolated from nuclear matrix-attached DNA using shotgun selection approaches.

The compact structural organization' of chromatin and the proper spatial orientation of individual chromosomes within a cell are partially provided by the nuclear matrix. The nuclear matrix is composed of DNA, RNA and proteins and WO 01/93836 PCT/US01/18657 serves as the site of DNA replication, gene transcription, DNA repair, and chromosomal attachment in the nucleus. Diverse sets of DNA sequences have been found associated with nuclear matrices and is referred to as matrix attachment regions or MARs. The MARs serve many functions, acting as activators of gene transcription, silencers of gene expression, insulators of transcriptional activity, nuclear retention signals and origins of DNA replication. Current studies indicate that different subsets of MARs are found in different tissue types and may assist in regulating the specific functions of cells. The presence of this complex assortment of structural and regulatory molecules in the matrix, as wcll as the in situ localization of DNA replication and transcription complexes to the matrix strongly suggest that the nuclear matrix plays a fundamental, unique role in nuclear processes. The structuring of genomes into domains has a functional significance. The inclusion of specific MAR elements within gene transfer vectors could have utility in many experimental and gene therapy applications. Many gene therapy applications require specific expression of one or more genes in targeted cell types for prolonged time periods. MARs within vectors could enhance transcription of the introduced transgene, prolong the retention of that sequence within the nucleus or insulate expression of that transgene from the expression of a cotransduced gene (reviewed by Boulikas, 1995; Bode et al, 1996).

Various biochemical procedures have been used to identify regulatory regions within genes. Traditionally, identification and selection of regulatory DNA sequences depend on tedious procedures such as transcription factor footprinting in vitro or in vivo, or subcloning of smaller fragments from larger genomic DNA sequences upstream of reporter genes. These methods have been used primarily to identify regions proximal to the 5' end of genes. However, in many instances, regulatory regions are found at considerable distances from the proximal 5' end of the gene, and confer cell type- or developmental stage- specificity. For example, studies from the groups of Grosveld and Engel (Lakshmanan et al., 1999) have shown that over 625 kb of genomic sequences surrounding the GATA-3 locus are required for the correct developmental expression of the gene in transgenic mice.

Extensive DNA stretches at distances 5-20 kb upstream of the gene were found to be responsible for the central nervous system-specificity of expression. The region WO 01/93836 PCT/US01/18657 between 20 to 130 kb upstream of the gene harbored regulatory regions for urogenital-specific expression ofGATA-3, whereas sequences 90-180 kb downstream of the gene conferred endocardial-specific expression.

The presently disclosed method has the potential of rapidly identifying regulatory control regions. In cells, chromatin loops are formed and different attachment regions are used in different cell types or stages of development to modulate the expression of a gene. The presently disclosed method for isolating regulatory regions based on their attachment to the nuclear matrix can identify regulatory regions irrespective of their distance from the gene. Although the human genome project is expected to be almost complete by the year 2000, information on the location and nature of the vast majority of the estimated 500,000 regulatory regions will not be available.

Example 1 Plasmid DNA condenses with various agents, as well as various formulations of cationic liposomes. The condensation affects the level of expression of the reporter beta-galactosidase gene after transfection of K562 human erythroleukemia cell cultures. Liposome compositions are shown in the Table below and in FIG. 2.

All lipids were from Avanti Polar Lipids (700 Industrial Park Drive, Alabaster, AL 35007). The optimal ratio of lipid to DNA was 7 nmoles total lipid/ig DNA. The transfection reagent (10 tg DNA mixed with 70 nmoles total lipid) was transferred to a small culture flask followed by the addition of 10 ml K562 cell culture (about 2 million cells total); mixing of cells with the transfection reagent was at 5-10 min after mixing DNA with liposomes. Cells were assayed for beta-galactosidase activity several times at 1-30 days post-transfection. The transfected cells were maintained in cell culture as normal cell cultures.

Best results were obtained when the cells used for transfection were at low number, not near confluence. In all experiments the transfection material was added directly in the presence of serum and antibiotics without removal of the transfection reagent or washings of the cells. This simplifies the transfection procedure and is suitable for lymphoid and other type of cell cultures that do not attach to the dish, but grow in suspension. All DNA condensing agents were purchased from Sigma. They WO 01/93836 PCT/US01/18657 were suspended at 0.1 mg/ml in water. Plasmid pCMV3 was purchased from Clontech and was purified using the Anaconda kit ofAlthea Technologies (San Diego, CA). PolyK is polylysine, mw 9,400. PolyR is polyarginine. PolyH is polyhistidine.

To 100 Rl plasmid solution (10 .tg total plasmid DNA) 20 pl or 50 pl of polyK, polyR, polyH, were added; the volume was adjusted to 250 pl with water followed by addition of about 70 pl liposomes (7 nmoles /tg DNA). After incubation for 10 min to 1 h at 20°C the transfection mixture was brought in contact with the cell culture. The best DNA condensing reagent was polyhistidine compared with the popular polylysine. The best cationic lipid was DC-cholesterol (DC-CHOL: 3 [N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol). SFV is Semliki Forest virus expressing beta-galactosidase. The results are shown in FIG. 2.

Liposome Molecular weight Composition Preparation L2 DDAB mw 631 DDAB 4.2 pmoles/ml 15 mg DDAB DOPE mw 744 DOPE 4.2 gmoles/ml 0.88 ml 20 mg/ml

DOPE

L3 DOGS-NTA mw 1015.4 DOGS-NTA 1 pmole/ml 5 mg DOGS DOPE 1 pmole/ml 0.185 ml DOPE L4 DC-Chol (mw 537) DC-Chol 1 pmole/ml 0.106 ml DC-Chol DOPE (mw 744) DOPE 1 pmole/ml mg/ml) 0.185 ml DOPE mg/ml) DOTAP (mw 698) DOTAP 1.4 pmole/ml 0.5 ml 10 mg/ml DOTAP DOPE (mw 744) DOPE 1.3 4mole/ml 0.25 ml DOPE mg/ml) L6 DODAP (mw 648) DODAP 1.54 4moles/ml 0.5 ml 10 mg/ml DOPE 1.3 imole/ml DODAP=5 mg=7.72 gmoles 0.25 ml DOPE mgiml) Example 2 Targeting Genes to Tumors Using Gene Vehicles (Lipogenes).

As shown in FIG. 3, tumor targeting in SCID (severe combined immunodeficient) mice were implanted subcutaneously, at two sites, with human MCF-7 breast cancer cells. The cells were allowed to develop into large, measurable solid tumors at about 30 days post-inoculation. Mice were injected intraperitoneously with 0.2 mg plasmid pCMVP DNA (size of the plasmid is ~4 kb) WO 01/93836 PCT/US01/18657 per animal carrying the bacterial beta-galactosidase reporter gene. Plasmid DNA (200[tg, 2.0 mg/ml, 0.1 ml) was incubated for 5 min with 200pl neutral liposomes of the composition 40% cholesterol, 20% dioleoylphosphatidylethanolamine(DOPE), 12% palmitoyloleoylphosphatidylcholine (POPC), 10% hydrogenated soy phosphatidylcholine (HSPC), 10% distearoylphosphatidylethanolamine (DSPE), sphingomyelin and 3% derivatized vesicle-forming lipid M-PEG-DSPE.

At this stage, weak complexation ofplasmid DNA with neutral (zwitterionic) liposomes takes place. This ensures homogeneous distribution ofplasmid DNA to liposomes at the subsequent step of addition of cationic liposomes. After complexation of plasmid DNA with zwitterionic liposomes, 50 P1l of cationic liposomes (DC-Chol 1 p mole/ml:DOPE 1.4 pmole/nl) were added and incubated at room temperature for 10 min. At this stage, a mixed liposome population is present and, most likely, formation of a type of liposome-DNA complexes containing lipids from the zwitterionic and cationic lipids takes place. The material was injected (0.35 ml total volume) to the intraperitoneal cavity of the animal. At 5 days post-injection the animal was sacrificed, the skin was removed and the carcass was incubated into X-gal staining solution for about 30 min at 37°C. The animal was incubated in fixative in X-gal staining for about 30 min (addition of 100 p1 concentrated glutaraldehyde to 30 ml X-gal staining solution) and the incubation in staining solution continued. Photos were taken in a time course during the incubation period revealing the preferred organs where beta-galactosidase expression took place.

Because of the tumor vasculature targeting shown in FIG. 3E, the data imply that transfer of the genes of angiostatin, endostatin, or oncostatin to the tumors (whose gene products restrict vascular growth and inhibit blood supply to the tumor) is expected to be a rational approach for cancer treatment. Also, a combination therapy using anticancer lipogenes with encapsulated drugs into tumor targeting liposomes appears as a rational cancer therapy.

It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and the following examples are intended to illustrate and not limit the scope of the invention.

Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

WO 01/93836 PCT/US01/18657 Table 3 Simple NLS Signal oligopeptide Protein and features PKKKRKV (SEQ ID NO:20) Wild-type SV40 large T protein A point mutation converting lysine-128 (double underlined) to threonine results in the retention of large T in the cytoplasm. Transfer of this peptide to the N-terminus of p-galactosidase or pyruvate kinase at the gene level and microinjection ofplasmids into Vero cells showed nuclear location of chimeric proteins.

PKKKRMV (SEQ ID NO:21) SV40 large T with a K--M change. Site-directed mutagenesis only slightly impaired nuclear import of large T.

PKKKRKVEDP (SEQ ID Synthetic NLS peptide from SV40 large T antigen crosslinked to BSA NO:22) or IgG mediated their nuclear localization after microinjection in Xenopus oocytes. The PKKGSKKA from Xenopus H2B was ineffective and PKTKRKV was less effective.

CGYGPKKKRKVGG (SEQ ID Synthetic peptide from SV40 large T antigen conjugated to various NO:23) proteins and microinjected into the cytoplasm of TC-7 cells. Specified nuclear localization up to protein sizes of 465 kD (ferritin). IgM of 970 kD and with an estimated radius of 25-40 nm was retained in the cytoplasm.

CYDDEATADSQHSTPPKKK SV40 large T protein long NLS. The long NLS but not the short NLS, RKVEDPKDFESELLS was able to localize the bulky IgM (970 kD) into the nucleus.

(SEQ ID NO:24) Mutagenesis at the four possible sites of phosphorylation (double underlined) impaired nuclear import.

CGGPKKKRKVG SV40 large T protein. This synthetic peptide crosslinked to chicken (SEQ ID NO:25) serum albumin and microinjected into HeLa cells caused nuclear localization.

PKKKIKV (SEQ ID NO:26) A mutated version of SV40 large T NLS. Effective NLS.

MKx 1CRLKKLKCSKEKPKC Yeast GAL4 (99 kD). Fusions of the GAL4 gene portion encoding the AKCLKx 5 Rx 3 KTKR (SEQ ID 74 N-terminal amino acid with E. coli p-galactosidase introduced into NO:27) yeast cells specify nuclear localization.

74 N-terminal amino acid MKx 11 CRLKKLKCSKEKPKC Yeast GAL4. Acted as an efficient nuclear localization sequence when A (SEQ ID NO:28) fused to invertase but not to p-galactosidase introduced by 29 N-terminal amino acid transformation into yeast cells.

PKKARED (SEQ ID NO:29) Polyoma large T protein. Identified by fusion with pyruvate kinase VSRKRPR (SEQ ID NO:30) cDNA and microinjection of Vero African green monkey cells.

Mutually independent NLS. Can exert cooperative effects.

CGYGVSRKRPRPG Polyoma virus large T protein. This synthetic peptide crosslinked to (SEQ ID NO:31) chicken serum albumin and microinjected into HeLa cells caused nuclear localization.

APTKRKGS SV40 VP1 capsid polypeptide (46 kD). NLS (N terminus) determined (SEQ ID NO:32) by infection of monkey kidney cells with a fusion construct containing the 5' terminal portion of SV40 VP1 gene and the complete cDNA sequence ofpoliovirus capsid VPI replacing the VPI gene of APKRKSGVSKC (1-11) Polyoma virus major capsid protein VP1 (11 N-terminal amino acid).

(SEQ ID NO:33) Yeast expression vectors coding for 17 N-terminal amino acid of VP1 fused to 0-galactosidase gave a protein that was transported to the nucleus in yeast cells. Subtractive constructs of VP1 lacking A1 to C11 were cytoplasmic. This, FITC-labeled, synthetic peptide crosslinked to BSA or IgG, caused nuclear import after microinjection into 3T6 cells.

Replacement of K 3 with T did not.

WO 01/93836 PCT/US01/18657 Signal oligopeptide Protein and features PNKKKRK (SEQ ID NO:34) SV40 VP2 capsid protein (39 kD). The 3' end of the SV40 VP2-VP3 (amino acid position 317-323) genes containing this peptide when fused to poliovirus VP I capsid protein at the gene level resulted in nuclear import of the hybrid VP1 in simian cells infected with the hybrid EEDGPQKKKRRL (307-318) Polyoma virus capsid protein VP2. A construct having truncated VP2 (SEQ ID NO:35) lacking the 307-318 peptide transfected into COS-7 cells showed cytoplasmic retention of VP2. The 307-318 peptide crosslinked to BSA or IgG specified nuclear import following their microinjection into NIH 3T6 cells.

GKKRSKA (SEQ ID NO:36) Yeast histone H2B. This peptide specified nuclear import when fused to p-galactosidase.

KRPRP (SEQ ID NO:37) Adenovirus Ela. This pentapeptide, when linked to the C-terminus of E. coli galactokinase, was sufficient to direct its nuclear accumulation after microinjection in Vero monkey cells.

CGGLSSKRPRP (SEQ ID Adenovirus type 2/5 Ela. This synthetic peptide crosslinked to chicken NO:38) bovine albumin and microinjected into HeLa cells caused nuclear localization.

LVRKKRKTE

3 SP (NLS 1) Xenopus N1 (590 amino acid). Abundant in X. laevis oocytes, forming (SEQ ID NO:39) complexes with histones H3, H4 via two acidic domains each LKDKDAKKSKQE (NLS2) containing 21 and 9 respectively. The NLS 1 is required but not (SEQ ID NO:40) sufficient for nuclear accumulation of protein N1. NLS 1 and 2 are contiguous at the C-terminus.

GNKAKRQRST v-Rel or p59 v-rel the transforming protein, product of the v-rel (SEQ ID NO:41) oncogene of the avian reticuloendotheliosis retrovirus strain T (Rev-T).

v-Rel NLS added to the normally cytoplasmic P-galactosidase directed that protein to the nucleus.

PFLDRLRRDQK NS1 protein of influenza A virus, that accumulates in nuclei of virus- (SEQ ID NO:42) infected cells. Determined to be an NLS by deletion mutagenesis of PKQKRKMAR NS 1 in recombinant SV40. The 1st NLS is conserved among all NS 1 (SEQ ID NO:43) proteins of influenza A viruses.

SVTKKRKLE (SEQ ID NO:44) Human lamin A. Dimerization of lamin A was proposed to give a complex with two NLSs that was transported more efficiently.

SASKRRRLE Xenopus lamin A. NLS inferred from its similarity to human lamin A (SEQ ID NO:45) NLS.

TKGKRKRID Xenopus lamin LI NLS inferred from its sequence similarity to (SEQ ID NO:46) human lamin A NLS.

CVRTTKGKRKRIDV Xenopus lamin LI. This synthetic peptide crosslinked to chicken (SEQ ID NO:47) bovine albumin and microinjected into HeLa cells caused nuclear localization.

ACIDKRVKLD Human c-myc oncoprotcin. This synthetic peptide crosslinked to (SEQ ID NO:48) chicken bovine albumin and microinjected into HeLa cells caused nuclear localization.

ACIDKRVKLD Human c-myc oncoprotein. Conjugation of the Ml peptide to human (SEQ ID NO:49) serum albumin and microinjection of Vero cells gives complete (M1, fully potent NLS) nuclear accumulation. M2 gave slower and only partial nuclear localization.

RQRRNELKRSP

(SEQ ID (M2, medium potency NLS) SALIKKKKKMAP Murine c-abl (IV) gene product. The pl60gag/v-abl has a cytoplasmic (SEQ ID NO:51) and plasma membrane localization, whereas the mouse type IV c-abl protein is largely nuclear.

WO 01/93836 PCT/US01/18657 Signal oligopeptide Protein and features PPKKRMRRRIE Adenovirus 5 DBP (DNA-binding protein) found in nuclei of infected (SEQ ID NO:52) cells and involved in virus replication and early and late gene PKKKKKRP (SEQ ID NO:53) expression. Both NLS are needed, and disruption of either site impaired nuclear localization of the 529 amino acid protein.

YRKCLQAGMNLEARKTKK Rat GR, glucocorticoid receptor (795 amino acid) NLS 1 determined by KIKGIQQATA (497-524 amino fusion with P-galactosidase (116 kD). NLS1 is 100% conserved acid) between human, mouse and rat GR. Whereas the 407-615 amino acid (SEQ ID NO:54) fragment of GR specifies nuclear location, the 407-740 amino acid fragment was cytoplasmic in the absence of hormone, indicating that sequence 615-740 may inhibit the nuclear location activity. A second (NLS2) is localized in an extensive 256 amino acid C-terminal domain. NLS 2 requires hormone binding for activity.

RKDRRGGRMLKHKRQRDD Human ER (estrogen receptor, 595 amino acid) NLS. NLS is between GEGRGEVGSAGDMRAMIN the hormone-binding and DNA-binding regions; ER, in contrast with O ACIDNLWPSPLMIKRSKK GR, lacks a second NLS. Can direct a fusion product with p- (amino acid 256-303) galactosidase to the nucleus.

(SEQ ID RKFKKFNK Rabbit PG (progesterone receptor). 100% homology in humans; F->L (SEQ ID NO:56) change in chickens. When this sequence was deleted, the receptor became cytoplasmic but could be shifted into the nucleus by addition of hormone; in this case the hormone mediated the dimerization of a mutant PG with a wild type PG molecule.

GKRKNKPK (SEQ ID NO:57) Chicken Etsl core NLS. Within a 77 amino acid C-terminal segment homologous to Ets2. When deleted by deletion mutagenesis at the gene level the mutant Etsl became cytoplasmic.

PLLKKIKQ (SEQ ID NO:58) c-myb gene product; directs puruvate kinase to the nucleus.

PPQKKIKS (SEQ ID NO:59) N-myc gene product; directs puruvate kinase to the nucleus.

PQPKKKP (SEQ ID NO:60) p53; directs puruvate kinase to the nucleus.

SKRVAKRKL c-erb-A gene product; directs puruvate kinase to the nucleus.

(SEQ ID NO:61) CGGLSSKRPRP Adenovirus type2/5 Ela. This synthetic peptide conjugated with a (SEQ ID NO:62) bifunctional crosslinker to chicken serum albumin (CSA) and microinjected into HeLa cells directed CSA to the nucleus.

MTGSKTRKHRGSGA Yeast ribosomal protein L29. Double-stranded oligonucleotides (SEQ ID NO:63) encoding the 7 amino acid peptides (underlined) and inserted at the N- MTGSKHRKHPGSGA terminus of the P-galactosidase gene resulted in nuclear import.

(SEQ ID NO:64) RHRKHP (SEQ ID NO:65) Mutated peptides derived from yeast L29 ribosomal protein NLS, KRRKHP (SEQ ID NO:66) found to be efficient NLS. The last two are less effective NLS, KYRKHP (SEQ ID NO:67) resulting in both nuclear and cytoplasmic location of P-galactosidase KHRRHP (SEQ ID NO:68) fusion protein.

KHKKHP (SEQ ID NO:69) RHLKHP (SEQ ID KHRKYP (SEQ ID NO:71) KHRQHP (SEQ ID NO:72) PETTVVRRRGRSPRRRTPSP Double NLS of hepatitis B virus core antigen. The two underlined RRRRSPRRRRQS (SEQ ID arginine clusters represent distinct and independent NLS. Mutagenesis NO:73) showed that the antigen fails to accumulate in the nucleus only when (One sequence, C-terminus) both NLS are simultaneously deleted or mutated.

ASKSRKRKL Viral Jun, a transcription factor of the AP-1 complex. Accumulates in (SEQ ID NO:74) nuclei most rapidly during G2 and slowly during G1 and S. The cell cycle dependence of viral but not of cellular Jun is due to a C->S mutation in NLS of viral Jun. This NLS conjugated to rabbit IgG can mediate cell cycle-dependent translocation.

WO 01/93836 PCT/US01/18657 Signal oligopeptide Protein and features GGLCSARLHRHALLAT Human T-cell leukemia virus Tax trans-activator protein. The most (SEQ ID NO:75) basic region within the 48 N-terminal segment. Missense mutations in this domain result in its cytoplasmic retention.

DTREKKKFLKRRLLRLDE Mouse nuclear Mxl protein (72 kD), Induced by interferons (among (604-620) 20 other proteins) Selectively inhibits influenza virus mRNA (SEQ ID NO:76) synthesis in the nucleus and virus multiplication. The cytoplasmic Mx2 has R->S and R-+E changes in this region.

CGYGPKKKRKV (SV40 large Synthetic peptides crosslinked to bovine serum albumin (BSA) and T) (SEQ ID NO:77) introduced into MCF 7 or HeLa S3 cells with viral co-internalization CGYGDRNKKKKE (human method using adenovirus serotype 3B induced nuclear import of BSA.

retinoic acid receptor) (SEQ ID NO:78)

CGYGARKTKKKIK

(human glucocorticoid receptor) (SEQ ID NO:79)

CGYGIRKDRRGGR

(human estrogen receptor) (SEQ ID

CGYGARKLKKLGN

(human androgen receptor) (SEQ ID NO:81) RKRQRALMLRQAR Human XPAC (xeroderma pigmentosum group A complementing 30-42 protein) involved in DNA excision repair. By site-directed (SEQ ID NO:82) mutagenesis and immunofluorescence. NLS is encoded by exon 1 which is not essential for DNA repair function.

EYLSRKGKLEL (SEQ ID T-DNA -linked VirD2 endonuclease of the Agrobacterium NO:83) tumefaciens tumor-inducing (Ti) plasmid. A fusion protein with P- (at the N-terminus) galactosidase is targeted to the nucleus. The T-plasmid integrates into plant nuclear DNA; VirD2 produces a site-specific nick for T integration. VirD2 also contains a bipartite NLS at its C-terminus (see Table 2).

KKSKKKRC (SEQ ID NO:84) Putative core NLS of yeast TRM1 (63 kD) that encodes the tRNA (95-102) modification enzyme N 2

N

2 -dimethylguanosine-specific tRNA methyltransferase. Localizes at the nuclear periphery. The 70-213 amino acid segment of TRM1 causes nuclear localization of Pgalactosidase fusion protein in yeast cells. Site-directed mutagenesis of the 95-102 peptide resulted in its cytoplasmic retention. TRM1 is both nuclear and mitochondrial. The 1-48 amino acid segment specifies mitochondrial import.

PQSRKKLR (SEQ ID NO:85) Max protein; specifically interacts with c-Myc protein. Fusion of 126- 151 segment of Max to chicken pyruvate kinase (PK) gene, including this putative NLS, followed by transfection of COS-1 cells and indirect immunofluorescence with anti-PK showed nuclear targeting.

QPQRYGGGRGRRW (SEQ ID Gag protein of human foamy retrovirus; a mutant that completely lacks NO:86) this box exhibits very little nuclear localization; binds DNA and RNA in vitro.

WO 01/93836 PCT/US01/18657 Table 4 "Bipartite" or "split" NLS Signal Oligopeptide Protein and features C-terminus Xenopus nucleoplasmin. Deletion analysis demonstrated the presence of a signal responsible for nuclear location.

TKKAGQAKKK (SEQ ID NO:87) Xenopus nucleoplasmin TKKAGQAKKKKLD Xenopus nucleoplasmin. Whereas these 17 amino acids had NLS (SEQ ID NO:88) activity, shorter versions of the 17 amino acid sequences were unable to locate pyruvate kinase to the nucleus.

TKKAGQAKKK(KLD) Xenopus nucleoplasmin. This 14 amino acid segment was (SEQ ID NO:89) identified as a minimal nuclear location sequence but was unable to locate puruvate kinase to the nucleus; three more amino acids at either end (shown in parenthesis) were needed.

CGQAKKKKLD Xenopus nucleoplasmin-derived synthetic peptide; crosslinked to (SEQ ID NO:90) chicken serum albumin and microinjected to HeLa cells specified nuclear localization. This suggests that nucleoplasmin may possess a simple NLS.

KRPAMINO ACID Xenopus nucleoplasmin bipartite NLS. Two clusters of basic TKKAGQAKKKK (SEQ ID NO:91) amino acids (underlined) separated by 10 amino acid are half NLS components.

HRKYEAPRHx 6 PRKR (SEQ ID Yeast L3 ribosomal protein (387 amino acid) N-terminal 21 NO:92) amino acid. Possible bipartite NLS. (Ribosomal proteins are transported to the nucleus to assemble with nascent rRNA).

Fusion genes with P-galactosidase were used to transform yeast cells followed by fluorescence staining with b-gal antibody. The 373 amino acid of L3 fused to p-gal failed to localize to the nucleus, unless a 8 amino acid bridge containing a proline was inserted between L3 and p-gal.

NKKKRKLSRGSSQKTKGTSASAK SV40 Vp3 structural protein. (35 amino acid C-terminus). By AR-IKRRNRSSRS (one sequence) DEAE-dextran-mediated transfection of TC7 cells with mutated (SEQ ID NO:93) constructs.

RVTIRTVRVRRPPKGKHRK Simian sarcoma virus v-sis gene product (p28Sis). The cellular (SEQ ID NO:94) counterpart c-sis gene encodes a precursor of the PDGF B-chain (platelet-derived growth factor). The NLS is 100% conserved between v-sis gene product and PDGF. This protein is normally transported across the ER; introduction of a charged amino acid within the hydrophobic signal peptide results in a mutant protein that is translocated into the nucleus. Puruvate kinase-NLS fusion product is transported less efficiently than cytoplasmic v-sis mutant proteins to the nucleus.

KRKIEEPEPEPKKAK Putative bipartite NLS of Xenopus laevis protein factor xnf7.

(SEQ ID NO:95) Inferred by similarity to the bipartite NLS of nucleoplasmin.

During oocyte maturation xnf7 is cytoplasmic until mid-blastulagastrula stage due to high phosphorylation. Partial dephosphorylation results in nuclear accumulation.

KKYENVVIKRSPRKRGRPRKD Yeast SWI5 gene product, a transcription factor. Underlined (SEQ ID NO:96)- basic amino acid show similarity to bipartite NLS of Xenopus nucleoplasmin. The SWI5 gene is transcribed during S, G2 and M phases, during which the SWI5 protein remains cytoplasmic due to phosphorylation by CDC28-dependent histone H1 kinase at three serine residues two near and one (double underlined) in the NLS. Translocated at the end ofanaphase/G1 due to dephosphorylation ofNLS. NLS confers cell cycle-regulated nuclear import of SWI5-p-galactosidase fusion protein.

WO 01/93836 PCT/US01/18657 Signal Oligopeptide Protein and features MKRKRNS 735-741 Bipartite NLS of influenza virus polymerase basic protein 2 (SEQ ID NO:97) (PB2). Mutational analysis of PB2 and transfection of BHK cells GIESIDNVMGMIGILPDMTPSTEM showed that both regions are involved in nuclear import.

SMRGVRISKMGVDETSSAEKIV Deletion of 449-495 region gives perinuclear localization to the 449-495 (SEQ ID NO:98) cytoplasmic side.

AHRARRLH (SEQ ID NO:99) "Tripartite" or "doubly bipartite" NLS of adenovirus DNA 6-13 (BSI) polymerase (AdPol). BSI and II functioned interdependently as PPRRRVRQQPP (SEQ ID NO:100) an NLS for the nuclear targeting of AdPol, for which BSIII was 23-33 (BSII) dispensable. BSII-III was more efficient NLS than BSI-II.

PARARRRRAP (SEQ ID NO:101) 39-48 (BSIII) KRKx 1 1 KKKSKK 207-226 Human poly(ADP-ribose) polymerase (116 kD). The linear (SEQ ID NO: 102) distance between the two basic clusters is not crucial for NLS activity in this bipartite NLS. Lysine 222 (double underlined) is an essential NLS component. DNA binding and poly(ADPribosyl)ating active site are independent of NLS.

GRKRAFHGDDPFGEGPPDKKGD Herpes simplex virus ICP8 protein (infected-cell protein). This (SEQ ID NO: 103) C-terminal portion of ICP8 introduced into pyruvate kinase (PK) caused nuclear targeting in transfected Vero cells. Inclusion of additional ICP8 regions to PK led to inhibition of nuclear localization.

KRPREDDDGEPSERKRARDDR Bipartite NLS of VirD2 endonuclease of rhizogenes strains of (SEQ ID NO:104) Agrobacterium tumefaciens. Within the C-terminal 34 amino acid. Each region (underlined) independently directs 3glucuronidase to the nucleus, but both motifs are necessary for maximum efficiency. VirD2 is tightly bound to the 5' end of the single stranded DNA transfer intermediate T-strand transferred from Agrobacterium to the plant cell genome.

WO 01/93836 PCT/US01/18657 Table 5. "Nonpositive NLS" lacking clusters of arginines/lysines Signal oligopeptide Protein and features QLVWMACNSAMIN Influenza virus nucleoprotein The underlined region O (327-345) when fused to chimpanzee al-globin at the cDNA level and ACIDFEDLRVLSFIRGTKVS microinjected into Xenopus oocytes specifies nuclear localization.

PRG 327-356 (SEQ ID NO:105) MNKIPIKDLLNPQ Yeast MAT a2 repressor protein, containing a homeodomain.

(NLS1 at N-terminus) (SEQ ID The two NLS are distinct, each capable of targeting P-galactosidase to NO: 106) the nucleus. However, deletion of NLS2 results in a2 accumulation at VRILESWFAKNIEN the pores. NLS1 and 2 may act at different steps in a localization PYLDT (NLS2 at amino acid pathway. Part of the homeodomain mediates nuclear localization in 141-159, part of the addition to DNA binding. The core pentapeptide containing proline and homeodomain) two other hydrophobic amino acids flanked by lysines or arginines (SEQ ID NO:107) (underlined) was suggested as one type of NLS core.

Rx 7 Kx 15 KIPRx 3 HFY Drosophila HP1 (206 amino acids) that binds to EERLSWYSDNED (SEQ ID heterochromatin and is involved in gene silencing. NLS identified by 3- NO:108) galactosidase/HP1 fusion proteins introduced by P-element mediated 152-206 (C-terminal transformation into Drosophila embryos.

segment) FVx7- Adenovirus type 5 E1A internal, developmentally-regulated 2 0 MxSLxYMx 4 MF NLS. This NLS functions in Xenopus oocytes but not in somatic cells.

This NLS can be utilized up to the early neurula stage.

WO 01/93836 PCT/US01/18657 Table 6. Nucleolar localization signals (NoLS) Signal oligopeptide Protein and features MPKTRRRPRRSQRKRPPTP Nucleolus localization signal in amino terminus of human p27 x (SEQ ID NO:109) III protein (also called Rex) of T cell leukemia virus type I (HTLV-I). When this peptide is fused to N-terminus of Pgalactosidase, directs it to the nucleolus. Deletion of residues 2- 8 (underlined), 12-18 (double-underline) or substitution of the central RR (dotted-underlined) with TT abolish nucleolar localization. Other amino acids between positions 20-80 increase nucleolar localization efficiency.

RLPVRRRRRRVP (SEQ ID NO:110) Adenovirus pTP1 and pTP2 (preterminal proteins, 80 kD) between amino acid residues 362-373. The 140 kD DNA polymerase of adenovirus when it has lost its own NLS can enter the nucleus via its interaction with pTP. The staining was nuclear and nucleolar with some perinuclear staining as well.

The NLS fused to the N-terminus of E. coli 3-galactosidase was functional in nuclear targeting.

GRKKRRQRRRP HIV (human immunodeficiency virus) Tat protein; localizes (SEQ ID NO:111) pyruvate kinase to the nucleolus. Tat is constitutively nucleolar.

RKKRRQRRR(AHQ) Tat positive trans-activator protein of HIV-1 (human Nucleolar localization signal immunodeficiency virus type The 3 amino acids shown in (SEQ ID NO:112) parenthesis are essential for the localization of the Pgalactosidase to the nucleolus. The 9 amino acid basic region is able to localize P-gal to the nucleus but not to the nucleolus.

KRVKLDQRRRP (SEQ ID NO: 113) Artificial sequence from c-Myc and HIV Tat NLSs that effectively localizes pyruvate kinase to the nucleolus.

FKRKHKKDISQNKRAVRR Human HSP70 (heat shock protein of 70 kD); localizes pyruvate (SEQ ID NO:114) kinase to the nucleus and nucleolus. HSP70 is physiologically cytoplasmic but with heat-shock HSP70 redistributes to the nucleoli, suggesting that the nucleolar targeting sequence is cryptic at physiological temperature and is revealed under heatshock.

RQARRNRRRRWRERQR (35-50) HIV-1 Rev protein (116 amino acid, nucleolar). Mutations in (SEQ ID NO:115) either of the two regions of arginine clusters severely impair nuclear localization. P-galactosidase fused to R4W was targeted to the nucleus, and fused to the entire 35-50 region, was targeted to the nucleolus.

RQARRNRRRRWRERQRQ (35-51) HIV-1 Rev protein. A fusion of this Rev peptide with 3- (SEQ D NO: 116) galactosidase became nuclear but not nucleolar. The 1-59 amino acid segment of Rev fused to P-galactosidase localized entirely within the nucleolus. Whereas the NRRRRW (bold) is responsible for nuclear targeting, the RR and WRERQRQ (double underlined) specify nucleolar localization. Rev may function to export HIV structural mRNAs from the nucleus to the cytoplasm.

WO 01/93836 WO 0193836PCT/USOI/18657 Table 7. Karyophilic clusters on non-membrane protein kinases Karyophilic peptides Non-membrane Species Features protein inase 73 FVVHKRCHE Protein kinase C (673 Bovine, human Known to translocate to the (SEQ ID NO: 117) aa) J3type nucleus following treatment of 96 DDPRSKHKFKIH cells with mitogens.

(SEQ ID NO: 118) 577 TKEiPGKRLG (SEQ ID NO: 119) 71 FVVHIRRCHEF Protein kinase C (697 bovine, humani y (SEQ ID NO: 120) aa.) type DDPRINKiHKFRLH (SEQ ID NO: 121) 591 TKIHPAIKRLG (SEQ ID NO: 122) 72 FVVHKRCHE Protein kinase C (673 rabbit type a and (SEQ ID NO: 123) aa)p 96 DDPRSKIIKFKIH (SEQ ID NO: 124) 577 TKHPGKRIG (SEQ ID NO: 71 FVVHRRCHE PKC-I (701 aa) rat brain (SEQ ID NO: 126)

DDPRNKHKFRLH

(SEQ ID NO: 127) 594 TKJIPGKRLG (SEQ ID NO: 128) 22 GENKMKSRIRKG Protein kinase C Drosophila 14 exons, 20 kb; 3 transcripts i (not conserved) (639aa, 75 kDa) adult flies; not expressed in 0-3h (SEQ ID NO: 129) Drosophila embryos; the SOSYVVHKRCHEYVT VVHKRCHE (SEQ ID (conserved) NO: I 33)motif (or VVHRRCHE (SEQ ID NO: 130) (SEQ ID NO: 134)) is conserved 211 PDDKDQSKKKTR among all PKC known.

TIK (not conserved) (SEQ ID NO: 13 1) 614PPFKPKIKHRKMC P (not conserved) (SEQ ID NO: 148 KKVLQDKRFK Glycogen synthase rat brain Phosphorylates glycogen synthase, NRELQIMRKLD (SEQ kinase 3 c-Jun, c-Myb; two isoforms ID NO: 13 5) GSK-3a encoded by discrete genes; highly (483 an) expressed in brain; both a and 1 forms are cytosolic hut also GSK-313 associated with the plasma (420 aa) membrane consistent with their role in signal transduction from the cell surface.

LQDRRFKNRELQ Zw3 Drosophila Product of the segment polarity (SEQ ID NO: 13 6) zeste-white 3 gene zw3; the protein encoded has 34% homology to cdc2; mutations in zw3 give embryos that lack most of the ventral denticles, differentiated structures derived from the most anterior region of ech segment.

WO 01/93836 PCT/US01/18657 Karyophilic peptides Non-membrane Species Features protein kinase 289ECLKKFNARRKL Ca /calmodulin- rat brain Composed of nine 50 kDa a- KGAIL dependent protein subunits and three 60 kDa p- (SEQ ID NO:137) kinase II (CaM kinase subunits; both are catalytic; II) p subunit (542aa, calmodulin- and ATP-binding 60.3 kDa) domains; highly expressed in forebrain neurons, concentrated in postsynaptic densities; acts as a 2+ Ca -triggered switch and could be involved in long-lasting changes in synapses.

290LKKFNARRKL CaM kinase II (478 rat brain This particular isoform is KGAILTTM (SEQ ID aa, 54 kDa) exclusively expressed in the brain; NO:138) a-subunit high enzyme levels in specific 450EETRVWHRRDGK brain areas; might be involved in (SEQ ID NO:139) short- and long-term responses to transient stimuli.

185 GFAKRVKGRT CADPK catalytic bovine (cardiac By Edman degradation of protein WTLCG subunit (349 aa, 40.6 muscle) fragments; mediates the action of (SEQ ID NO: 140) kDa) and is activated by cAMP; consists of two regulatory and two catalytic subunits; cAMP releases the C subunit from the inactive R2C2 cADPK; two cDNAs were cloned encoding two isoforms of the catalytic subunit of cADPK in mouse.

186 GFAKRVKGRTW CADPK bovine cDNA was isolated by screening a TLCG (catalytic subunit) bovine pituitary cDNA library; (SEQ ID NO:141) (350 aa) 93% sequence similarity to known bovine cADPK; represents the second gene for the catalytic subunit of cADPK.

29 EEEIQELKRKLH CGDPK (SEQ ID bovine lung By protein sequencing; composed KCQSVLP (SEQ ID NO:144) of two identical subunits activated NO: 142) (670 aa, 76.3 kDa) in an allosteric manner by binding 389 KILKKRHIVDTR of cGMP and not by dissociation (SEQ ID NO: 143) of catalytic subunit as in cADPK; sequence similar to cADPK 117 KTLKKHTIVK TPK3 S. cerevisiae cAMP-DPK is a tetrameric protein (SEQ ID NO: 145) (398 aa) with two catalytic and two cADPK regulatory subunits; cAMP activates the kinase by dissociating the catalytic subunits from the tetramer; all three TPK 1, 2, 3 are catalytic subunits.

16S 2

H

13 GHG2 SNF1 (633aa, 72 kDa) S. cerevisiae Ser/Thr kinase; 166 EYCHRHKIVHRD autophosphorylated; plays a LKP (SEQ ID NO:146) central role is carbon catabolite 495 PLVTKKSKTRWH repression in yeast required for FG (SEQ ID NO:147) expression of glucose-repressible genes; region 60-250 shows high sequence similarity to cAMPdependent protein kinase (cADPK).

WO 01/93836 WO 0193836PCT/US01/18657 Karyophilic peptides Non-membrane Species Features protein kinase PVKKKKIKREIK Casein kinase 11 Drosophila CKII is composed of a and (SEQ ID NO: 148) subunit, catalytic) nzelanogaster subunits in a a2132 130-150 kDa 269 DILQRHSRKRW (336aa) protein; the a-subunit is the ERF (SEQ ID NO: 149) catalytic and the j3 is 146 PKSSRHHHTDG CKII (P-subunit, Drosophila autophosphorylated.

(SEQ ID NO: 150) regulatory) (2l5aa) melanogaster 142 PKSSRI{HHTDG CKII (1-subunit, bovine (lung) (SEQ ID NO: 15 1) regulatory) (209aa, 24.2 kDa) 108 PKQRHRKSLG KINI (1064 aa, 117 S. cei-evisiae 30% aa similarity to bovine (SEQ ID NO: 152) kDa) cADPK and 27% (KINi1) or 129 GSMCKVKLAK (KJN2) an similarity to v-Src HRYTNE within the kinase domain; the (SEQ ID NO: 153) catalytic domains of KINI and 506 DRKIIAKIRNQ KJN2 are near the N-terminus and (SEQ ID NO: 154) are structural mosaics with features 638 GNIFPKLSQRR characteristic of both Tyr and KKTIEQ Ser/Thr kinases.

(SEQ ID NO: 155) 773 PPLNVAKGRKL HP (SEQ ID NO: 156) 87 ELRQFHRRSLG KIN2 (1152 aa, 126 S. cerevisiae (SEQ ID NO: 157) kDa) 111 GKVKJVKHRQ TKE (SEQ ID NO:158) 217 GSLKEHHARKF ARG (SEQ ID NO: 159) 807 LSVPKGRKLHP (SEQ ID NO: 160) STE7 (515 an) S. cerevisiae Implicated in the control of the (SEQ ID NO: 16 1) three cell types in yeast: a and 472 PSKDDKFRI{WC ala) of which a and a. cells are RKIKSKIKEDKRIKRE haploid and are specialized for (SEQ ID NO: 162) mating whereas a/a cells are diploid and are specialized for mejosis and sporulation; with the exception of the mating type locus, MAT, all cells contain the same DNA sequences. STE7 gene produces insensitivity to celldivision arrest induced by the yeast mating* hormone, a-factor.

722 QRRVKKLPSTTL S6KIla (733 an) Xenopus (SEQ ID NO: 163) QRRVKKLPSITL S6KII 13 Xenopus (SEQ ID NO: 164) 742 QRRVKKLPSITL S6K11 (752 aa) Chicken (SEQ ID NO: 165)111 713QRRVRKLPSTTL S6KII (724aa) Mouse (SEQ ID NO: 166)111 WO 01/93836 WO 0193836PCT/US01/18657 Karyophilic peptides Non-membrane Species Features protein kinase 1 6GV'VYKGRIIKTTG CDC2Hs Human Isolated by expressing a human (SEQ ID NO: 167) (297aa) cDNA library in S. pombe and 120 FCHSRRVLHRD p 34 cdc2 selecting for clones that LKP (SEQ ID NO:168) complement a mutation in the cdc2 yeast gene; the human GDC2 gene can complement both the inviability of a null allele of S.

cerevisiae CDC28 and cdc2 mutants of S. poinbe; CDC2 mP.NA appears after that of CDK2.

GVVYKARIHKLSGR cdc2 (297aa) S. pomibe High homology to S. cerevisiae (SEQ ID NO: 169) CDC28.

11 9HSHRVLHRIJLKP CDK2 (cell division Human The human CDK2 protein has (SEQ ID NO: 170) kinase 2) (298 aa) sequence identity to human p 3 4 cdc2 and 89% sequence identity to Xenopus EglI kinase;, human CDK2 was able to complement the inviability of a null allele of S. cerevisiae CDC28 but not cdc2 mutants in S. pombe.

CDK2 mRNA appears in late Gl/early S.

109 FCHSHRVLHRD Eg 1 (297aa) Xenopus Cdk2-related LKIP (SEQ ID NO: 17 1) 125 GIAYCHSHRILH CDC28 (298a) S. cerevisiae The homolog of S. pombe Cdc2

RDLKP

(SEQ ID NO: 172) 119 HSHRVIHRDLKP cdk3 (305aa) Human (SEQ ID NO: 173) 56 KELKHKNIVR PSSALRE (291 aa) Human cdc2 -related kinase.

(SEQ ID NO: 174) (SEQ ID NO: 175) 1 MDRMKKIKRQ PCTAIRE- 1 (496 aa) Human cdc2-related kinase.

ternus) (SEQ ID NO: 176) 141 DKPLSRRLRRV (SEQ ID NO: 177) I MKKFKRR PCTAIRE-2 (523 aa) Human cdc2 related kinase.

(SEQ ID NO: 178) 129 RNRIHRRIS (SEQ ID NO: 179) 172 SRRSRRAS (SEQ ID NO: 180) 304 HRRKVLHR (SEQ ID NO: 18 1) 512 GHGKNRRQSM LF (SEQ ID NO: 182) 163 HTRKILHR PCTAIRE-3 Human cdc2 related kinase.

(SEQ ID NO: 183) (380 aa) 369 PGRGKNRkQSIF (SEQ ID NO: 184) WO 01/93836 WO 0193836PCT/USOI/18657 Karyophulic peptides Non-membrane Species Features protein kinase 69 EVFRRKRRLH KKIALRE (358 aa) Human cdc2-related kinase.

(SEQ ID NO: 185) (SEQ ID NO: 187) 302 DKPTRKTLRKSR KHH (SEQ ID NO: 186) 1 MVKEIJKNT nirn1+ gene product S. pombhe (SEQ ID NO: 188) (new inducer of 87 DGELFHYIREIHGP nmitosis); protein (SEQ ID NO: 189) kinase (370 aa) 114 DAVAHCHRFRFR HRD (SEQ ID NO:l190) 295 KKSSSKKVVRRL

QQRDD

(SEQ ID NO: 19 1) 194 PAQKLRKKNNFD Wee 1+ gene product S. ponibe The Weel+ gene functions as a (SEQ ID NO: 192) (877aa) dose-depenident inhibitor that 388 KQI{RPRKNTNFT delays the initiation of mitosis PLPP (SEQ ID NO: 193) until the yeast cell has attained a 592 KYAVKKLKVKF certain size; Wee 1 has a protein SGP (SEQ ID NO: 194) kinase consensus probably cdc2 kinase.

266 PNETRIRIKRAN CDC7 (497 aa) S. cerevisrzae Required for mnitotic but not RAG (SEQ ID NO: 195) meiotic DNA replication presumably to phosphorylate specific replication protein factors; implicated in DNA repair and meiotic recombination; some homology with CDC28 and oncogene protein kinases but differs in a large region within the phosphorylation receptor domain.

4SYDHVRKTRVAIK ERKl (MAP kinase) Rat Known to translocate to the (SEQ ID NO: 196) (367 an; 42 kDa) nucleus following their activation by phosphorylation at T- 190, and Y-192 (T-183, Y-185 in ERK2).

59 ILKHFKHE FUS3 (353aa) S. cerevisiae MAP-(ERK1)-related.

(SEQ ID NO: 197) 252 QIKSKRAKEY KSSI (368 aa) S. cerevisiae MAP-(ERK1)-related.

(SEQ ID NO: 198) ELVKIHLVKHGSN SWI6 S. cerevisiae Activator of CACGA-box with (SEQ ID NO: 199) (803aa, 90kDa) sequence similarity to cdcl0; GKAKKIRSQLL required at START of cell cycle.

(SEQ ID NO:200) EQRLKRURIDVS1JED cdcl0 S. pornbe (SEQ ID NO:201)

SNIKSKCRRVV

(SEQ ID NO:202) 37 PPKRIRTD CTD kinase (528 aa) S. cerevisiae Consists of 3 subunits of 58, 38, (suggested by the 58 kfla subunit and 32 kDa; disruption of the 58 authors) (SEQ ID (catalytic) kI~a gene gives cells that lack CTD NO:203) kinase, grow slowly, are cold 492 KLARKQKRP sensitive, but hnve different (SEQ ID NO:204) phosphorylated forms of RNA pol WO 01/93836 PCT/US01/18657 Karyophilic peptides Non-membrane Species Features protein kinase 29 GVSSVVRRCIHKP Phosphorylase kinase Rabbit (skeletal (SEQ ID NO:205) (catalytic subunit) muscle) (386aa) 489 KKYMARRKW Myosin light chain Chicken gizzard Ca2+/calmodulin-activated; QKTGHAV kinase (MLCK) (669 phosphorylated by cADPK; first (SEQ ID NO:206) aa) described as responsible for the phosphorylation of a specific class of myosin light chains; required for initiation of contraction in smooth muscle.

314 PWLNNLAEKAK Myosin light chain Rabbit (skeletal By protein sequencing.

RCNRRLKSQ kinase (partial 368 muscle) (SEQ ID NO:207) carboxy-terminal aa 334 ILLKKYLMKRR sequence)

WKKNFIAVS

(SEQ ID NO:208) 28 GVSSVVRRCIHKP Phosphorylase kinase Mouse (muscle) Glycogenolytic regulatory enzyme; (SEQ ID NO:209) (PhK) (catalytic y undergoes complex regulation; subunit) (389 aa) composed of 16 subunits containing equimolar ratios of a, 3, y and 6 subunits; high levels in skeletal muscle; isoforms in cardiac muscle and liver; cDNA probe does not hybridize to X chromosome in mice and is thus distinct from the mutant recessive PhK deficiency that results in glycogen storage disease.

WO 01/93836 WO 0193836PCT/USOI/18657 Table 8. Nuclear localization signals on DNA repair proteins Putative NLS Gene Equivalent protein Features product in other species HIGHER EUKARYOTES None ERCC1 RADIO 297aa; L)BD; interacts (N-terminus) strongly with ERCC4 (XPF) MDPGKDKEGvpqpsgppaRKKF to form an excision (bipartite NLS) endonuclease; unless the (SEQ ID NO:210) KDKxi 1RKK is a bipartite NLS it may depend upon its binding with ERCC4 for its nuclear import.

None ERCC2 RAD3 cer) 760 aa; DNA helicase 681DKRFARGDKRGKLPR (XPD) component of IFIIH, (near the C-terminus) (four essential for cell viability; positive one negative over a contains one nucleotideheptapeptide stretch) binding, one DNA-binding, (SEQ ID NO:21 1) and seven domains characteristic of helicases; 52% identity with S. cer RAD3 at the ammno acid level.

S DRDKKXSRKRI{YEDEE ERCC3 SSL2 (S cer) 782 aa; helicase, component (SEQ TD NO:2 12) (XPB) Haywire(Dros) of TIIH essential for cell 522 YVAIKTKKRILLYTM viability; helix-turn-helix, (SEQ ID NO:213) DNA-BD, and helicase (weak NLS if at all, hydrophobic domains environment) 769 PSKIIVHPLFKPYRK (SEQ ID NO:2 14) 84 KKQTLVKRRQRKD ERCC5 RAD2; 1186 aa in human, 1196 in X.

(SEQ ID NO:215) (XPG) Rad13 laevis; 3'incision 210 EFTKRR1RTL endonuclease; involved in (SEQ ID NO:2 16) homologous recombination; 390 DESMIKDRKDRLP strongly nuclear (SEQ ID NO:217) 1170 GKKRRXLRRARGRK RKT (SEQ ID NO:2 IS) 253PQKQEKKPRKIMLNEASG ERCC6 RAD26 1493aa; involved in the (SEQ ID NO:219) CS-B preferential repair of active 314 PNKKARVLSKKEERLKK genes; nonessential for cell HIKYXLQKR (SEQ ID NO:220) viability 406 PLPKGGRQLKVP (SEQ ID NO:221) 455 DGDEDYYKORLRRWNK

LRLQDKEKRLKLEDDSEESD

(SEQ ID NO:222) 1028 DVQTPKCHLKRRIQP

X

8 PKRKKFP (SEQ ID NO:223) 1180 KHKSKTKHHSVAEEETL

EKHILRPKQKPKX

1 5

PHLVKK

RRY (SEQ ID NO:224) 1324 PAGKKSRFGKKRN (SEQ ID NO:225) WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Gene Equivalent protein Features product in other species 21 PASVRASIERJgQRALM XPA RAD14 273 aa; zinc finger domain; LRGAR (SEQ ID NO:226) involved in lesion 160 PPLKFIVKKNPHHiSQW recognition GD (weak) (SEQ ID NO:227) 2 10 NREKMKQKKFDKKVKE (weak because of F) (SEQ ID NO :228) 72 YLRRAMKRFN (weak) XPC RAD4 (23% identity, 823 aas, 92.9 kDa; very (SEQ ID NO:229) 44% similarity) hydfrophilic protein; might be 262 PSAKGKP.NKGGRKXRSK involved in lesion PSSSEEDEGPG (SEQ ID recognition since XPC cells NO:230) (40% of all XP cases) can 297 QRRPHGRERR (weak) repair active parts of the (SEQ ID NO:23 1) genome whereas inactive and 368 RTHJ(GSHRKDP (weak) the nontranscribed strand of (SEQ ID ND:232) active genes are not repaired 384 SSSSSSSKRGKKMCSDG (SEQ ID NO:233) 531 ALKRHLLKYE (weak) (SEQ ID NO:234) 594 SNRARXARLAEP (SEQ ID NO:235) 660 PNLHRVARKLD (weak) (SEQ ED NO:236) 716 ERLKEKEKKEKR (SEQ ID NO:237) 740 TRRKRRYG (SEQ ID NO:238) 801 GGPKKTKRERJK (SEQ ID NO:239) KSKAKSKARREEEEED XPC 940 aa; the first 117 aa are (SEQ ID NO:240) lacking in the Legerski and 54 GKR.KRG (SEQ ID NO:241) Peterson, (1992) XPC 69 GPAKKKVAKVTVK sequence (see above); the (SEQ iD NO:242) following 823aa are 103 PSDLKKAHHLKRG identical.

(SEQ ID NO:243) 82 EIDRRKKRPLENDGPVKK Rep-3 Swi4 (S porn) 1 13 7aa; mismatch repair KVKKVQQKE (SEQ ID (mouse) protein; Rep-3 is in the NO:244) Duc-1 immediate 5' flanking region 375 KENVRDKKKG (HeLa) of DHFR gene (89 bp) but (SEQ ID NO:245) transcribed from the opposite 571 FGRRKLKKWT strand; a bidirectional (SEQ ID NO:246) promoter is used for both 710 PLLKXRKDELQG transcripts.

(SEQ ID NO:247) 1091 KELEGLINTKRKRLKYF AKLW (SEQ ID NO:248) 422 EKJIEGKHQKLL (weak) hMSH2 MSH2 (S cer) human mismatch repair (SEQ ID NO:249) protein; homologous to S.

cerevisiae MSH2; associated with the hereditary nonpolyposis colon cancer on chromosome 2p 1 6 WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Gene Equivalent protein Features product in other species 397 PDIRRLTYKLMRG MSH2 (SEQ ID NO:250) (S cer) 547 DAKELRKHKKYIE (SEQ ID NO:251) 869 VKMAKRKANE (SEQ ID NO:252) GELAKRSERRAEAE Human Rad2 Rad2 porn) 400 aa; iequired for fidelity (SEQ ID NO:253) of chromosome separation at 354 KRKEPEPKGSTKXKAK mitosis; limited similarity to TG (SEQ ID NO:254) RAD2 (ssDNA nuclease), 394 GKFKRGK (SEQ ID rad13, and XPG NO:255) None mouse 339 aa; reconmbination-repair 1 protein; 83% homology to S cerevisiae RAD5 1 and homology to E. coli RecA.

None HHR23B RAD23 Subunit of XPC (125 kDa) None HHR23A RAD23 Subunit of XPC (125 kDa) 32 PSQAEKXSRARAQ RPA (34 kDa RPA (70, 34, and 14 kDa (SEQ ID NO:256) subunit) subunits) might stabilize the helicase-melted DNA around the lesion; antibodies against RPA 32 kDa subunit inhibit DNA replication.

GAKKRKIDDA ATPase QI RecQ coli) 649 aa; altered in XPC cells; (SEQ ID NO:257) undetermined role in repair PKKIPRGKM (SEQ ID NO:258) HMG-l Calf thymus HMG 1 EHKKK-HP (SEQ ID NO:259) (259 aa); involved in the ETKIKKFKDP (SEQ ID NO:260) recognition of cisplatin

EKSKKKK(E(D)

4 1 (SEQ ID lesions NO:26 1) E3G2KKKKKFAK (SEQ ID NO:262) 512 RDEIKIKLKKAKAK SSRP1 ABF (S cer) 709 aa, 81 kDa, structure- MAKDRKSRKKP (SEQ ID specific recognition protein NO:263) 1; involved in recognition of 619 GESSKRDKSKKKKKVKV cisplatin-induced lesions; KMEKK (SEQ ID NO:264) also involved in Ig gene 674 GENKSKKKRRRSEDSEE recombination; one HMG- EE (SEQ ID NO:265) box, similarity to SRY, MTFII, LEF- 1, TCF- Ia, and I MPKRGKKG (SEQ ID Ref-i Redox factor I from HeLa NO:266) (HAPi) cells; 37 kDa, 318 aa; apurinic/apyrimidinic (AP) endonuclease for DNA repair but also of redox activity stimulating Jun/Fos DNA binding.

I MPKRcIKKc3 HAPI Exoll 323 aa; apurinic/apyrimidinic (SEQ ID NO:267) (bovine) coli) (AP)-endonuclease ExoA (S, WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Gene Equivalent protein Features product in other species

DROSOPHILA

1 MGPPKKSRKI)RSGGDKF Haywire ERCC3 (XPB) helicase with 66% identity to GKKRRGQDE human ERCC3; flies (SEQ ID NO:268) SSL2 (S cer) expressing marginal levels of EMSYSRKRQRFLVNQG Haywire display motor (weak) (SEQ ID NO:269) defects and reduced life span

YYEHRKKNJGSVHPLFK

KFRG (bipartite) (SEQ ID NO:270) 77 ARGKXKQPK (SEQ ID Rrpl HAP1 Recombination repair protein NO:27 1) 679 aa; the 252 aa C- 98 KPKGRAKKA (SEQ ID terminal domain is NO:272) homologous to AP- 157 QAKGRKKKELP (SEQ ID endonucleases, whereas the NO:273) 1-426 aa domain is highly 179 EPPKQRARKE (SEQ ID charged, carries all of the NO:274) putative NLSs.

241 PPKAASKRAKKGK (SEQ ID NO:275) 282 PKKRAKKTT (SEQ ID NO:276) 317 EPAPGKKQKKSAD (SEQ ID NO:277) 336 EEEAKPSTEThPAKGR KKAP (SEQ ID NO:278) 372 KPARGRKKA (SEQ ID NO:279) 394 GSK~rKKAKKAE (SEQ ID NO:280) S. CEREVISIAE 200 IEKIRRKLYISGG RAD1 ERCC4 1100 aa; 30% sequence (SEQ ID N0:28 1) (XPF) identity to Radl16; RAD 1 515 NKKRGVRQVLLN (SEQ Radl6 interacts strongly with ID N0:282) RADIO 565 KEQVTTKRRRTRG (conserved in Rad 16) (SEQ ID NO:283) 1024 NLRKKIKSFNKLQ (SEQ ID NO:284 89 RQRKE-RRQGKRE RAD2 XPGC 1031 as, 117.8 kDa; ssDNA (SEQ ID NO:285) Radl3 endonuclease; rad mutants 907 ENKFEKDLRKKLVNNE are defective in incision (SEQ ID NO:286) 984 RDVNKRKKKGKQKRI (SEQ ID NO:287) 1017 KRISTATGKLKKRKM (SEQ ID 672 GKDDYGVMVLADRR1 RAD3 ERCC2 or XPD; 778 aa, 89,779 Da; SRKRSQLP (contains the bulky cer) Rad 15 or Rhap3 sequence identity to rad16; F) (SEQ ID NO:2 89) ATP-dependcnt DNA helicase; single-stranded _________DNA-dependent ATPase.

WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Gene Equivalent protein Features product in other species 26 PLSRRRRVRRKNQPLPD RAD4 XPC 754 aa; mutations in RAD4 AKKIKFKTG (SEQ ID NO:290) that that inactivate the 134 NEERKRRIKYFHMLYL excision repair function of (SEQ ID NO:291) RAD4 result in truncated 160 EW17NSKP.LSRKLSNL proteins missing the C- (weak) (SEQ ID NO:292) terminal one-third of RAD4.

254 EMSANNKRK 7

KTLKRSD

weak (SEQ ID NO:293) 382 WMvNSKVRKRRITKDDF GEK (SEQ ID NO:294) 403 RKVITALHHRKRTKID DYED (SEQ ID NO:295) 504 KTGSRCKXVIKRTVGR-P (SEQ ID NO:296) 150 FHPKRRPIYGFR (SEQ ID RAD5 1169 aa; helicase involved in NO:297) postreplication-repair (RAD6 215 DSRGRKI{ASM (SEQ ID epistasis group); binds DNA NO:298) with the seven helicase 297 DGESLMKRRRTEGGNK motifs and with zinc fingers; REK (SEQ ID NO:299) increases the instability of 1152 DEDERRKRIE3 poly (GT) repeats in the yeast (SEQ ID NO:300) geniorne.

I MSTPARRRLMRDFKRM RAD6 RAD6 mediates the KEDAPP (SEQ ID NO:301) ubiquitmnation of H2A and H12B3 histones GVAKiLRKEKSGAD RADiO ERCCI 210 aa; forms an (SEQ ID NO:302) endonuclease with RAD I, 76 DDYNRKRPFRSTRPGK the basic and tyrosine-rich (SEQ ID NO:303) central domain was suggested to bind DNA by ionic interactions and intercalation.

172 EGKAHRREKKYE RAD14 XPAC 247aa, 29.3 kDa; two zinc (SEQ ID NO:304) fingers; involved in lesion 200 NRLRE-KKH1GKAHIHH recognition; 27% sequence (SEQ ID NO:305) identity and 54% sequence similarity (if conserved residues are grouped together) to human XPA; deletion of RAD 14 gene generates high UV sensitivity.

345 ERRKQLI(KQGPKRP Ixrl 591 aa; two consecutive (SEQ ID NO:306) cer) HMG boxes; involved in 479 ETYKKRIKEWESCYPDE recognition of 1,2-intrastrand (SEQ ID, NO:307) d(GpG) and d(ApG) cisplatin crosslinks.

None RAD23 HHR23 WO 01/93836 WO 0193836PCT/US01/18657 Putative NLS Gene Equivalent protein Features product in other species 483 LTGKKLKTIINRIILSG RAD26 ERCC6 1075aa; disruption of the weak (SEQ ID NO:308) (yeast CS-B (hum) RAD26 gene gives viable 934 NALRKSRKKITKQYEIGT ERCC6) yeast cells unable to

PX

9 GEIRKRDP preferentially repair the (SEQ ID NO:309) actively transcribed strands; surprisingly, in contrast to human CS-B cells, disruption of the RAD26 in yeast does not cause sensitivity to UV, Cisplatin, or X-rays.

634 KPTSKPKRVRTATKKKIP MREI I Rad32 (S porn) mnelotic recombination (SEQ IDNO:310) protein; functions in the 408 FYKiKRSPVTRSKKSG same pathway with RAD5 1 (SEQ ID NO:31 1) none; RAD5 1 RecA coli) 402 aa; essential for repair of 361 GFKKGKGCQR DSBs and recombination; (SEQ ID NO:312) associates strongly with RAD52; self associates; neither RAD51I nor RAD52 possess a typical simple

NLS.

none; RAD51 364 aa 328 GFKKGKGCQR lactis) (SEQ ID none; RAD52 Rad22 504 aa; rad52 mutants are 155 ERAKKSAVTDALKRSLR defective in ionizing GFGXgDKDFLAKIDKVKFDP radiation, mitotic PD (tripartite) recombination, mating-type (SEQ ID NO:3 14) switching, and repair of 1 MARRRLPDRPP RADS4 898 aa; recombination-repair (SEQ ID NO:315) protein; ATP-binding motif; GGRSLRKRSA helicase domains; in the (SEQ 1D NO:3 16) same subfamily of helicases 99 QLTKRRKD with MOTl1 and SNF2.

(SEQ ID NO:3 269 DETVFVKSKRVKASSS RAD55 Similarity to RecA, and (extremely weak if at all NLS) lower similarity to RAD51, (SEQ ID NO:3 18) RADS7, and DMC I 317 GEDRKREGRNLKR (SEQ ID NO:3 19) 371 PISRQSKKRKFDYRVP RAD57 460 aa; nucleotide-binding (SEQ ID NO:320) domain; limited similarity to 1 62 GLKKPRKKTKSSRH SSL2 ERCC3 (XPB) 843 aa; putative helicase that (SEQ ID NO:32 1) seems to function in repair 688 GRILRAKRRN'DEG but also in the removal of (SEQ ID NO:3 22) secondary structures in the 784 GRGSNGHKRFKS (weak) untranslated region of inRNA (SEQ ID NO:323) to allow ribosome binding scanning.

WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Gene Equivalent protein Features product in other species TRRHLCKIKGLSE (weak) DMC1 RecA 334 aa; yeast homolog of (SEQ D NO324)RecA, meiosis-specific; 277 GRKPGGH12RK7RGdmcl mutants are defective DER bipatite (SE IDin reciprocal recombination NO:325)and accumulate DSBs 11 ETEKR.CKQKEQRY PMS1 904 aa, 103 kDa; mismatch- (SEQ ID NO:3 26) repair protein; MutL (Salmonella) and HexB homolog None H1RR25 Hhpl, HhplI (S porn) Mutations in HRR25 Ser/Thr 1 MIDLRVGRKFRIGRKIG CKI (marin protein kinase cause defects (SEQ ID NO:3 27) in DNA repair and 139 GRRGXgGLSKIKYRDFNT retardation in cell cycling IRHIP (Bipartite weak NLS) (SEQ ID 96 HELTKRSSRRNVETEK YKL5 10 383 aa; structure-specific (SEQ ID NO:329) endonuclease; two domains of about 100 aa with sequence similarity to N- and C-terminal regions of RAD2.

200 MLAMLARRKKKMSAK MOTI Modifier of transcription 1; (SEQ ID NO:330) 1867 aa; DNA helicase of S.

617 EHYKVKHTEK (weak cerevisiae required for NLS) (SEQ ID NO:331) viability; increases gene 670 LHPEKK-RSISE (weak expression of several., but NLS) (SEQ ID NO:332) not all, pheromoneresponsive genes in the absence of STEI12; the 1257 to 1825 aa domain (568 aa residues) has homology to and RAD54 S. POMB3E SSIDEx 5 SIKRKRRI (SEQ ID Swi4 Duc-l 113 kDa; KCII sites are NO:333) Rep-3 upstream of NLS like in large T; the homologous prokaryotic MutS and HexA lack NLS 96 GELAKRVAR.HQKARE Rad2 380 aa (weak NLS) (SEQ ID NO:334) 362 GSAKRKRDS (SEQ ID NO:335) 372 KGGESKKKR (SEQ ID NO:336) None Rad9 -427 aa; no homology to other DNA repair proteins; rad9 fission yeast mutants are sensitive to both. UV and ionizing radiation; may be involved in recombinationrepair.

None Rhp3 or ERCC2 772 aa; DNA helicase; 681 DKRYGRSDKRTKLPK radl15 RAD3 identity to RAD3 and (SEQ IT) NO:337) identity to ERCC2; essential viability WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Gene Equivalent protein Features product in other snecies 464 PPSKRRRVRGG Rad16 RADI Function in repair of UV (SEQ ID NO:33 8) damage for both cyclobutane dimer and photoproduct lesions; Radi 6 interacts w ith Swi 431 DFKQAILRKRKNESPE Rad2l 628 aa, 67.8 kDa, acidic EVEP (SEQ ID NO:339) protein; a single base substitution in mutant rad2 1 changing an lie into a Thr, is responsible for the low efficiency in repair of DSBs after -radiation although capable of arresting Q2.

490 DKKAKKG (SEQ ID Rad22 RAD52 496 aa; functions in NO:340) recombination-repair and matin-tvpe switchin2.

394 DVVQFYLKKKYTRSKRN Rad32 MREI 1(S cer) 648 aa; meiotic DG (weak because of Y) (SEQ recombination protein; rad32 ID NO:341) mutants are sensitive tog- 575 PSPALLKKTNKRRELP and UV radiation; functions (SEQ ID NO:342) in the same pathway with Rhp)51 (RAD5 1).

I recombination-revair GLAKKYRDHKTHLHIP (weak Hhpl CKI (mamnm) Ser/Thr protein kinase; NLS because of Y and H) (SEQ HRR25 (S cer) mutation in this gene causes ID NO:343) repair defects None Hhp2 CKI (mamm) Ser,'Thr protein kinase; GLAKCKYRD' KTHMHP (H in HRR25 (S cer) mutation in this gene causes HhplI is replaced by F in Hihp2) repair defects (SEQ ID NO:344 WO 01/93836 WO 0193836PCT/UStil/18657 Table 9. NLS in Transcription factors NLS and Flanks Protein factor and features highly basic HR4QRTRK7R (SEQ ID Human GCF (OC-factor) NO:345) LRRKSRP (SEQ ID NO:346) SRRTKRRQ (SEQ ID NO:347) GRKRKKRT (SEQ ID NO:348) Oct-6 protein transcription factor from mouse cells GRRRIKKRT (SEQ ID NO:349) Mouse Oct-2 protein transcription factors (Oct-2.I for Oct-2.6 isoforms) ARKIRKRT (SEQ ID NO:350) Oct-3 from mouse P19 embryonal carcinoma cells NRRQKGKRS (SEQ ID 1) ECRP.KKKE Human ATF-I. In basic region/leucine zipper.

(SEQ ID NO:352) ERKKRRRE (SEQ ID NO:3 53) Human ATF-3 (in basic region that binds DNA) AKCRINKKKEKT (SEQ ID NO:354) SKKXIRL (SEQ ID NO:355) Mousc Prr.1 (Friend erythroleukemia cells). Related to ets oncogene QKGNRK-KM (SEQ ID NO:356) VKKVKKKL (SEQ ID NO:357) VKRKKI (SEQ ID NO:358) Human PRDII-BFl1 that binds to IFN-P gene promoter. (The largest CRLNRYRKLE (SEQ ID DNA-binding protein known, of 298 kD).

NO:359) IRKRRKMK (SEQ ID NO:360) PKXKRLRL (SEQ ID NO:361) GKKKKRKREKL Murine LEF-l1 (3 97 aa). Lymphoid-specific with an HMG I-like box.

(within the HMG-box) NLS is identical to that of human TCF- Ia.

(SEQ ID NO:362) GKKKKRKREKL Human TCF-la (399 an) (within the HMG-box) (T cell-specific transcription factor that activates the T cell receptor (SEQ ID NO:363) Ca). Contains an HMG box. NLS core is identical to that of murine LEF-1.

GKKKRRSREKH Human TCF-l (within the HMG-box) (SEQ ID (uniquely T cell-specific). HMG box containing.

NO:364) PKKCRARF (SEQ ID NO:365) FKQRRIKL (SEQ ID NO:366) Xenopus laevis Oct-l1(within POU-domain) INRRRKKRT (SEQ ID NO: 367) NRRQKEKRI (SEQ ID NO:3 68) DKIRSRKIRXRSK (SEQ ID Drosophila Suvar 7 gene product involved in position-effect NO:369) variegation (932 aas). Five widely spaced zinc-fingers could help RLRjIDRKRN (SEQ ID NO:3 70) condensation of the chromatin fiber.

AK-RSRRS (SEQ ID NO:37 1) WO 01/93836 WO 0193836PCT/UStil/18657 NLS and Flanks Protein factor and features IRKRRKMKSVGD2E 2 (SEQ ID Human MBP-l (class I MUC enhancer binding protein 1) mw 200 NO:372) kD. Induced by phorbol esters and mitogens in Jurkat T cells.

(not suggested as NLS by the authors; between the 1 st and 2nd zinc finger)

PPKKKR-LRLAE

(suggested as NLS by the authors; just before 2nd zinc finger) (SEQ ID NO:373)

CRNRYRKLE

(within I1st zinc finger) (SEQ ID PRIRKRRV (SEQ ID NO:375) rat TTF-1 (thyroid nuclear factor that binds to the promoter of HRYKMKRQ (SEQ ID NO:376) thyroid-specific genes). An homneodomain protein.

N (SEQ ID NO:377) Human thyroid hormone receptor a (c-erbA-l gene). Belongs to the DDSKRVAKRKL (SEQ ID family of cytoplasmic proteins that are receptors of hydrophobic NO:378) ligands such as steroids, vitD, retinoic acid, thyroid hormones. The NRERRRKEE (SEQ ID NO:3 79) ligand binding may expose the NLS for nuclear import of the WKQRRKF (SEQ ID NO:380) receptor-ligand complex.

NRRK.RKRS (SEQ ID NO: 38 1) Drosophila gcl (germ cell-less) gene product (569 aa, 65 kD), located PKKKKL (SEQ ID NO:3 82) in nuclei, required for germ line formation.

ARRKRRRL (SEQ ID NO:383) C. elegans Sdc-3 protein (sex-determining protein) (2,150 aas). A LKFKiKVRD (SEQ ID NO:384) zinc finger protein.

FK!KFRKF (SEQ ID NO:385) GKQKRRF (SEQ ID NO:386) ERLKRDKKRtE.KE (SEQ ID NO:387) TRGRPKKVKE (SEQ ID NO:388) SKKRGRRRKKT (SEQ ID NO:3 89) TRRQKRAKV (SEQ ID NO:390) SRKSKKRLRA (SEQ ID NO:391) LKXIRRKIKNKI (SEQ ID Drosophila BBF-2 (related to CREB/ATE) NO:392) ESRRKKKE (SEQ ID N:3 93) Group~ 0000 DRNKKKKE (SEQ ID NO:394) Xen op us RAR (retinoic acid receptor) ARRRRP (SEQ ID NO:395) GRRRRA (SEQ ID NO:396) Human ATF-2 (the 2nd and 3rd NLS are in basic region that binds DEKRRKV (SEQ ID NO:397) DNA) CRQKRKV (SEQ ID NO:398) ERKRRD (SEQ ID NO:399) Myn (murine homolog of Max). Forms a specific DNA-binding SRXKLRME (SEQ ID NO:400) complex with c-Myc oncoprotein through a helix-loop-helix/leucine EEKRKRTYE (SEQ ID NOA:01) humnan NFKB p 6 5 (550 aa).

Not binding DNA; conaplexed with p50 that binds DNA. NFKB p 50 contains a NLS (Table 3b).

WO 01/93836 WO 0193836PCT/USOI/18657 NLS and Flanks Protein factor and features GRRRRA (SEQ ID NO:402) Human HBl6, a cAMP response element-binding protein DEKRRKF (SEQ ID NO:403) SRCRQKRKV (SEQ ID NO:404) SKKKKTKV (SEQ ID NO :405) Human TFIIE-3 (general transcription initiation protein factor; forms NRPDKKKI (SEQ ID NO:406) tetramer U0A2 with TFILE-ct) QRRKKP (SEQ ID NO:407) QKKRRFKT (SEQ ID NO:408) SRKRKM (SEQ ID NO:409) Human kup transcriptional activator (433 aas). Two distantly spaced zinc fingers. Expressed in hematopoietic cells and testis.

ERKRIRN'RLA (SEQ ID Mouse Jun-B3 homologue to avian sarcoma virus 17 oncogene v-jun NO:410) product. One region is similar to yeast GCN4 and to Fos.

ATKCRKRKL (SEQ ID NO:41 1) (19 an stretch) DKRx6ERKRRD (N-terminus) Max (specifically associates with c-Myc, N-Myc, L-Myc). The Max- (SEQ ID NO:4 12) Myc complex binds to DNA; neither Max nor Myc alone exhibit QSRKKLRME (C-terminus) appreciable DNA binding.

(SEQ ID NO:4 13) DKEKKLKLEEDE (within an Chicken VBP (vitellogenin gene-binding protein). Leucine zipper.

acidic region) (SEQ ID NO:4 14) Related to rat DBP.

IKKAKKV (SEQ ID NO:415) TRRIKKN (SEQ ID NO:416) T 'RDDKRRA (SEQ ID NO:417) Xenopus borealis B I factor. Closely related to the mammalian USF.

EVERRRRDK (SEQ ID Binds to CACGTG in TFIIIA promoter to developmentally regulate NO:4 18) its expression.

TRDEKRRA (SEQ ID NO:419) Human USF (upstream stimulatory factor) activating the major late EVERIR k (SEiQ ID adenovirus promoter NO:420) YRRYPRRRG (SEQ ID YB-i, a protein that binds to the MHG class II Y box. YB-i is a NO:421) negative regulator.

QRRPYRRRUF (SEQ ID NO:422) YRPRFRRG (SEQ ID NO:423) QRRYRRN (SEQ ID NO:424) YRRRRP (SEQ ID NO:425) AKE-RQKKD(SEQ ID N24:426) Human TFEB Binds to IgH enhancer.

ERRRRF (SEQ ID NO:427) LKIER QKIK (SEQ ID NO:428) Human TFE3 (536 aa). Binds to pE3 enhancer of IgH genes.

I'ERRRRFN (SEQ ID NO:429) YFRRRRLEKD (SEQ ID NO:430) KTVALKRRKASSRL (SEQ ID Human DrI (176 aa, 19 kD). Interacts with TBP (TATA-bindmng NO:43 1) protein) thus inhibiting association of TFIIA and/or TFIIB with TBP.

TBP-Dri association is affected by DrlI phosphorylation to repress activated and basal transcription.

I LRRRGRQTY (SEQ ID Drosophila ultrabithorax protein (from the conserved 61 amino acid NO:432) homeodomain segment only). Conserved in the antenappedia 27 LTRRRRIEM (SEQ ID homeodomain protein.

NO :43 3) 51 QNRRMKLKKEI (SEQ ID NO:434) WO 01/93836 WO 0193836PCT/USOI/18657 NLS and Flanks Protein factor and features SNRRRPDHR (SEQ ID NO:435) C. elegans sex-determining Tra- I protein. Zinc finger. Peaks in the VYRGRRRVRRE (SEQ ID second larval stage.

NO:436) P7AP 2

RRRRSADNKD

2

(SEQ

ID NO:437) PKKPRJTQF (SEQ ID NO:438) EKRKKERN (SEQ ID NO:439) Yeast NPS1 transcription protein factor (1359 aa) involved in cell LLRRLKKEVE (SEQ ID growth control at G2 phase. Has a catalytic domain of protein NO:440) kinases.

EPLGRIRQKKRVy2D 2

(SEQ

ID NO:441)

(EDAIKKRREARERRRLRQ)

(SEQ ID NO:442)

DKETTASRSKRRSSRKKRT

(SEQ ID NO:443) ESKIKKXPKL (SEQ ID NO :444) KKTAAKKTKTKS (SEQ ID NO:445) QRKRQKL (SEQ ID NO:446) Human 243 transcriptional activator (968 aas), induced by mitogens KAKKQK (SEQ ID NO:447) in T cells. N-terminal half is homologous to oneoprotein Rel and LRP.KRQK (SEQ ID NO :448) Drosophila Dorsal protein involved in development. The C-terminal half contains repeats found in proteins involved in cell-cycle control of yeast and tissue differentiation in Drosophila.

RDIRRRGKMNiV (SEQ ID Mouse NF-E2 (45 lcD), an erythroid transcription factor from mouse NO:449) erythroleukemia (MIEL) cells. Involved in globin gene regulation.

QNCRKRKLE (SEQ ID Binds to AP-lI-like sites. Homology to Jun B, GCN4, Fos, ATFi1 and NO:450) CREB in basic region/leucine zipper (see Fig. 2).

Group 00exe0 DKIRRKN (SEQ ID NO:45 1) Human glucocorticoid receptor ARKTKKKI (SEQ ID NO :452) 473 DKARRKNCP (SEQ ID Mouse and human GR (glucocorticoid reeptor) NO:453) EARKTKKKIKGIQ (SEQ ID NO:454) Group 000xO YRVRRERN (SEQ 1D NO:455) C/EBP (CCAAT/enhancer binding protein).

VRkSRD SEQ ID NO:456) Functions in liver-specific gene expression.

DRLRKRVE (SEQ ID NO:457) DKIRRKN (SEQ ID NO:458) Human mineralocorticoid receptor ARKSKKL (SEQ ID NO :459) DKIRRKN (SEQ ID NO:460) Human PR (progesterone receptor) GRKFKKF (SEQ ID NO:46 1) EEVQRKRQKLMP (SEQ ID Human and mouse NFrB 105 kD precursor of p50 (968 aas) (first R NO:462) is at 361 position).

EEVQRKRQKL (SEQ ID Human NF-KB p50 (DNA-binding subunit). Identical to protein NO:463) KBF1, homologous to rel oncogene product. NF-KB p65 also contains a NLS (Table 3a).

rGKTRTRKQ (SEQ ID NO:464) Human TEF- I (SV40 transcriptional enhancer factor 426 aa.

ARRKSRD (SEQ U) NO:465) WO 01/93836 WO 0193836PCT/UStil/18657 NLS and Flanks Protein factor and features QRKE-RSKS (SEQ ID NO:466) Rat, mouse, human IRE-i (interferon regulatory factor-i). Induced in TKSKTKRKI (SEQ ID NO:467) lymphoma T cells by the pituitary peptide hormone prolactin.

Regulates the growth-inhibitory interferon genes.

GKCKKKNQ (SEQ ID NO:468) Ehirlich ascites S-Il transcription factor. A general factor that acts at the elongation step.

ERSKKRiSRE (SEQ ID NO:469) Tobacco TAF- 1 transcriptional activator ERELKREKRKQ (SEQ ID ARRSRLRKQ (SEQ ID NO:47 1) YKLDHMRRIETDE (SEQ ID Drosophila TFIIEa (433 aa), a general transcription factor for RNA NO:472) polymerase IL. Composed of subunits cc and P.

DKNR'RKS (SEQ ID NO:473) Human ER (estrogen receptor); 595 aa.

IRKD-RRG (SEQ ID NO:474) IKRSKKN (SEQ ID NO:475) EQRRIIRIE (SEQ ID NO :47 6) Yeast ADA2 (434 aa), a potential transcriptional adaptor required for TTRAEKKRIL (SEQ ID the function of certain acidic activation domains, NO:477) LDKKRSKEAKE (SEQ ID NO:478) EAALRRKIRTISK Yeast GCN5 gene product (439 aa), required for the function of (SEQ ID NO:479) GCN4 transcriptional activator and for the activity of the HAP2-3-4 complex.

Group OOxO NKKMRRNRF (SEQ ID Mouse LFB3 NO:480) NRRKx 4 RQK (SEQ ID NO:481) TKA(GRRNRF (SEQ ID Mouse LFB I NO:482) NRRKx 4 RI{K (SEQ ID NKKMRRNRFK (SEQ ID rat vHNF 1 -A NKKMRRNR (SEQ ID NO:485) murine HNF-lIP TKKGRRNRF (SEQ ID mouse HNF-lI NO:486) NKKMR1RNRF (SEQ ID human vILNFl NO:487) TKKGRRNRF (SEQ ID rat liver HNFlI NO:488) LRRQKRFK (SEQ ID NO:489) Tat HNF-3fP QQH3SH 4 Q (SEQ ID NO:490) LRRQKRFK (SEQ ID NO:49 1) rat IINF-3y LRRQKRFK (SEQ ID NO:492) rat HNF-3 a LKEKERKA (SEQ ID NO:493) rat DBP a protein factor that binds to the D site of the albumin gene MKKARKV (SEQ ID NO :494) promoter PRRE-RY (SEQ ID NO:495) rat AT-BP 1. Highly acidic domain. Two zinc fingers. Binds to the B3-domain of al~ -antitrypsin gene promoter and to the NF-icB site in te UH gene enhancer.

DRRVRXGKV (SEQ ID A 19 kD Drosophila melanogaster nonhistone associated with NO:496) heterocbromatin.

WO 01/93836 WO 0193836PCT/UStil/18657 NLS and Flanks Protein factor and features SKITGRR-ARRLDP (SEQ ID murine EBB (early B-cell factor) of 591 aa. Regulates the pre-B and NO:497) B lymphocyte-specific mb-I gene, Expressed in pre-B and B-cell GRRTRRE (SEQ ID NO:498) human Sp I DEQ gjA-E K-E (SEQ ID yeast SNF2, a transcriptional regulator of many genes.

NO:499) IRRIHKVIRP (SEQ TD NO:500) LLRRLKKDVE (SEQ ID NO:501) Group Ox6Elx9 AKAKAKKA (SEQ ID NO:502) mouse AGP/EBP (87% similarity to CIEB3P), ubiquitously expressed YKMRREN (SEQ ID NO:503) VRKSRD 'KA (SEQ ID NO:504) AKAKAKKA (SEQ ID NO:505) rat LAP, a 32-kD liver-enriched transcriptional activator, also present YKMRRERN (SEQ ID NO:506) in lung, with 71% sequence similarity to C/EBP. Leucine zipper.

VRk SRDK-A (SEQ ID NO:507) Accumulates to maximal levels around birth.

Y QRRE-(SEQ ID NO:508) Ig/EBP- 1 (immunoglobulin gene enhancer-binding protein), Forms VKKSRLKSKQK (SEQ ID heterodimers with C/EBP.

NO:509) EDPEKE-KRIKELE (SEQ ID mouse c-Myb NO:5160----- MRRKV (SEQ ID NO:51 1) DYYKVKRPKTD (SEQ ID Drosophila eyes absent protein (760 aa), a nuclear protein that 12) functions in early development to prevent programmed cell death and GRARGRRIIQ (SEQ ID to allow the event that generate the eye to proceed. Mutations cause 13) programmed cell death of eye progenitor cells.

FRYRKIKDIY (SEQ ID Group 0xex09 AKAKAKKA (SEQ ID NO: 515) rat IL-6DBP interacting with interleukin-6 responsive elements. Has a leucine zipper domain.

DKRQRNRC (SEQ ID NO:5 16) mouse H-2RIIBP (MHC class I genes H1-2 region 11 binding protein).

DKRQRNRG (SEQ ID NO: 517) protein factor from the thyroid/steroid hormone receptor family VKSKAKKT (SEQ ID NO: 518) human NF-1L6 (345 aa). Specifically binds to ILI 1-responsive YKIRERN SE D N: 519) element in the IL-6 gene. Leucine zipper. Homology to C/EBP.

QKKNRNKC (SEQ ID NO: 52 1) mouse PPAR (peroxisome proliferator activated receptor) Group eeexxBO EQIRKLVKKIIG (SEQ ID yeast RAPLI NO:522) It binds regulatory sites at yeast mating type silencers.

FRRSMKRKA (SEQ ID human vitamin D receptor (427 aa) NO:523) Group eoxxO WO 01/93836 PCT/USOI/18657 NLS and Flanks Protein factor and features LKRIHQRRH (SEQ ID NO:524) mouse WT1 (the murine homolog of human Wilms tumor predisposition gene WTI) LKRHQRRH (SEQ MD NO:525) human WT33 (Wilms' tumor predisposition) Group e9exxO LKESKRKYDE (SEQ ID yeast SW13 99 kD, highly acidic protein. Global transcription NO:526) activator.

EVLKVQKRRIYD (SEQ ID human RBAP-1 (retinoblastoma-associated protein 1) factor (412 aa).

NO:527) A protein that binds to the pocket (functional domain) of the retinoblastomna (RB) protein involved in suppression of cell growth (tumor suppressor). The transcription factor E2F, implicated in cell binds to the same pocket of RB.

WO 01/93836 WO 0193836PCT/USOI/18657 Table 10. NLS in other nuclear proteins Putative NLS Protein YXSKKKA (SEQ ID NO:528) Yeast L3 TKKLPRKT (SEQ ID NO:529) TRKKGGRRGRRL (SEQ ID NO:530) Yeast 59 ribosomal protein C-terminus ARATRRKRCKG (SEQ ID NO:53 1) Yeast Li 16 ribosomal protein GKGKYRNRRW (SEQ ID NO:532) yeast L2 ribosomal protein (homiologous to Xenopus LI). Encoded by intronless genes.

GKGKMRNRRRIQRRG (SEQ ID NO:533) Xenopus laevis, L ribosomal protein (homologous NKKVKRRE-LKKN-(SEQ ID NO:534) to yeast L2) Encoded by intronless genes.

AKTARRKA (SEQ ID NO:535) IKAKEKKP (SEQ ID NO :536) dKKAK (SE-Q ID NO:537) AKIAKIRQ (SEQ ID NO:538) ERKEKS (SEQ ID NO:539) human S6 ribosomnal Protein (homologous to yeast GKRPRTKA (SEQ ID NO: 540) SlO) BKRRRI (SEQ ID NO:541) LKKQRTICKNKE (SEQ ID NO:542) PKMRRRTYR (SEQ ID NO:543) Rat L17 ribosomal protein (184 aas) KXKISQKKLKK (SEQ ID NO:544) YMRRRTYRA (SEQ ID NO:545) Podocoryne camea (hydrozoan, Coelenteratumn) E\VK1VSKKKI (SEQ ID NO:546) L 17 ribosomnal protein (184 aas) highly homologous to rat LI 17.

ERINRKD-KDAKFR (SEQ ID NO:547) human, rat ribosomal S 13 protein ERKRKS (SEQ ID NO:548) yeast S 10 ribosomal protein (homologous to human QRIQRKRH (SEQ ID NO:549) S6) IRKRIRA (SEQ I-D NO:550)

GRRRKKLHRSRSRSRERRSRSRDRGRG

12 GRER 35 kD subunit of U2 small nuclear DRRRSRDRER (SEQ ID NO: 551) ribonucleoprotein auxiliary factor (U2AF), an essential mammalian splicing factor. U2AF 3 interacts with the 65 kb subunit (U2AF 65 Both proteins are concentrated in a small number of subnuclear organelles, the coiled bodies.

EFEDPRD (SEQ ID NO:5 52) human UsnRNP-associated 70 k protein (437 aas) ETREERME (SEQ ID NO:553) that is phosphorylated at Arg/Ser-rich domains; EAGDAPPDP (SEQ ID NO:554) involved in splicing EERMERiKRREK (SEQ ID NO:555) HRDRD RDRERERRESRERDKERE-lSSRD RRRRSRSRkEER.1-- RERSKDKDPDRXK-RRS SRSRERARRERERKEE (SEQ ID NO:556)

RDRDRERPR-SRERERRRDRDRDRDRDREH

.KRGER (SEQ ID NO:557) QKiRNNKKSKKKRCAE (SEQ ID NO:558) yeast TRM 1 enzyme for the N 2

,N

2 EKLRKLKI (near C-terminus) (SEQ ID NO:559) dimethylguanosine modification of both mitochondrial and cytoplasmic tRNAs. TRM1 is both nuclear and mitochondrial, The first motif is within a region (70-2 13 aa segment) known to cause nuclear localization of Ji-galactosidase.

NKRKRV (SEQ ID NO:560) Yeast nucleoporin NUJP (1076 an, 113 kD);1 an SLKNRSNRKRE (SEQ ID NO:5 61) integral component of the pore complex. Involved EPKRKIRRLP (SEQ ID NO:562) in both binding and translocation steps of nuclear ARMiRRSKR (C-terminus) (SEQ ID NO:563) import.

WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Protein KAEKEx 3 KVD2E 2 (SEQ ID NO:564) Chicken, Xenopus No 38 nucleolar (38 kD); Kx3Kx5Kx3R (SEQ ID NO:565) involved in intranuclear packaging of preribosomal particles. Shuttles between nucleus and cytoplasm.

KTEREAEKALEEKx 7 R (SEQ ID NO:566) Chicken, hamster nucleolin (92 kD). Binds Kx 5 Kx 7 Kx- 4 RX3EDTTEETLR (SEQ ID NO:567) preribosornal RNA. Shuttles between nucleus and RG2)RG2RG3RG2FG2RG3RGFG 2

RG

3

FRG

2

RG

4 cytoplasm.

DHI(PQOKKIKFE (SEQ ID NO:568) (C-terminus) WYKJIFKKTKD (SEQ ID NO:569) human SATBI. (763 aa) which binds selectively to AT-rich MARs with mixed A, T, C on one strand excluding G. Binds to minor groove with little contact with bases.

TKKKSFK (SEQ ID NO:57) yeast CC1, a ceruiomrm cutting ndotnues (KSERERLRSLEER (SE Q D NO:575) r5at nuclepori T5e orE epeat (139 is, 155rm KKEKKRKSED ka) aSE pDN:)ocrtein cofthe uleardn poeincomplex;cll EEKKKKSK (SQ I NO:73)containsn 4onsyteusies fo varou kin epassf cytop lai reio ofM peno C paret DKKRSKR((SEQ ID NO:57 7) yth EI cel c c iomcutn ndnces KP.RKLRDSKER (SEQ ID NO:57 Calf thus hisoine H4 rNp5 (30a 14-24in (10 cosnu stsfra)iu kns PAIRRLA (SEQ ID NO:57)hua 2vaindifrnalyepseduig 32-41 EHARRKT (SEQ ID NO:580) 74-80 ARIRGERA 127-135 (SEQ ID NO:581) Calf thymus H3 (135 aa) GSIIHKAKGK 121-129 (SEQ ID NO:582) Calf thymus H2A (129 aa) RGKSGKARTKAKSRSSR 3-19 (SEQ ID Sea urchin Psammnechinus miliaris H2A (123 aa) NO:583) PKKGSKKA 10-17 (SEQ ID NO:584) Calf thymus 112B QKIKDGKKRKRSRKES 22-36 (SEQ ID NO:585) (125 aa) GKRHRKRKGSY (SEQ ID NO:586) Sea urchin Psamnmechinus miliaris H2B (122 aa) 22-34 PRTDKKRRRKRKES 19-32 (SEQ ID NO:5 87) Starfish H2B (121 aa) PAKAPKKKA 12-20 (SEQ ID NO:588) Trout testis Hi EAKKPAKIKA 104-112 (SEQ ID NO:589) (194 aa) AKKPKKV 128-134 (SEQ ID NO:590) AKKSPKKAKKP 142-152 (SEQ ID NO:591) PKKVKKP 183-189 (SEQ ID NO:592) WO 01/93836 WO 0193836PCT/USOI/18657 Putative NLS Protein PRRKAKRA 30-37 (SEQ ID NO:593) PKKAKKT 11 9-125 (SEQ ID NO:594) AKAKKAKA 129-136 (SEQ ID NO:595) AKKARKAKA 139-147 (SEQ ID NO:596) AKKAKKPKKKA 171-181 (SEQ ID NO:597) AK-KAKKIPAKK 182-191 (SEQ ID NO:598) SPKKIAKKP 192-19 9 (SEQ ID NO:599) AKKSPKKKKAKRS 200-212 (SEQ ID NO:600) PKKAKKA 213-219 (SEQ ID NO:601) AKKAKKS 227-23 3 (SEQ ID NO:602) PRY-AGKRRSPKKARK 234-248 (SEQ ID NO:603) Sea urchin Parechinus angulosus sperm HI1 (248 aa) ARRRKTA 1-7 (SEQ ID NO: 604) Annelid sperm Hia IRKFIRKA 55-61 (SEQ ID NO:605) (119 aa) PKKKICA 83-88 (SEQ ID NO:606) AKKPKAKKVKKP 89- 100 (SEQ ID NO: 607) AKIKKTNRARKPKTKKNR 104-120 (SEQ ID NO:608) PKRiKVSS 1-7 (SEQ ID NO:609) Calf thymus HMG14 EEPKRRSARLS 14-24 (SEQ ID NO:610) (100 aa) PKRKAEGDAK 1 -10 (SEQ ID NO: 61 1) Calf thymus HMG17 PKGKIKGKA 52-59 (SEQ ID NO:612) (89aa; 9,247 D) PKKPRGKM (SEQ ID NO:613) Calf thymus HMG 1 EHKKKHP (SEQ ID NO: 614) (259 aa) EThKKKFKDP (SEQ ID NO:615)

EKSKI(KK(E/D)

4 1 (SEQ ID NO:616)

E

3 _GKKKKKFAK (SEQ ID NO:6 17) EHKIKJ-IP (SEQ ID NO: 618) Calf thymus H1MG 2 PKGDKKGKKKDP (SEQ ID NO:619) (256 aa) E493KKKXF AK (SEQ ID NO:620) PKRKSATKGDEPARR 1- 15 (SEQ ID NO:62 1) Trout testis H16 (60 aa) KPKKAAAPKKA 30-34 (SEQ ID NO:622) WO 01/93836 PCT/US01/18657 References U.S. Patent Documents 4,394,448 July, 1983 Szoka, Jr. et al.

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Claims (10)

1. A method for producing micelles with entrapped negatively charged polynucleotides, comprising: a) combining an effective amount of negatively charged polynucleotides c with a buffer solution containing from about 20% to about 80% ethanol to prepare a polynucleotide-ethanol solution; b) combining the polynucleotide-ethanol solution with an effective amount of cationic lipid and fusogenic-karyophilic peptide to form electrostatic micelle complexes wherein the negatively charged polynucleotides are neutralized by the cationic lipids and fusogenic-karyophilic peptides.

2. The method of claim 1, further comprising combining an effective amount of a negatively charged therapeutic agent in the buffer solution.

3. The method of claims 1 or 2, wherein the fusogenic-karyophilic peptide includes a signal peptide or a nuclear localization signal (NLS) peptide.

4. The method of any one of claims 1 to 3, further comprising reducing the ethanol comprising the micelles of step b) to below 10% ethanol. The method of any of claims 1 to 4, wherein the micelle complexes of step b) are combined with a fusogenic lipid in a molar ratio from about 1:1 to about 2:1.

6. A micelle containing entrapped negatively charged polynucleotide produced by the method of any one of claims

7. The method of any one of claims 1 to 5, further comprising combining the micelle complexes with an effective amount of liposome to produce liposome encapsulated micelles.

8. A liposome containing entrapped negatively charged polynucleotide produced by O the method of claim 7. O

9. A method for delivering a negatively charged polynucleotide to a subject, Scomprising administration to the subject of an effective amount of the micelle of claim 6. A method for delivering a negatively charged polynucleotide to a subject, c comprising administration to the subject of an effective amount of the liposome of claim 8.

11. Compositions obtained by the method of any one of claims 1-7.

12. A method for producing micelles with entrapped negatively charged polynucleotides according to claim 1, substantially as herein described with reference to the Examples and Drawings.