US 2003OO72794A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0072794A1 Boulikas (43) Pub. Date: Apr. 17, 2003

(54) ENCAPSULATION OF PLASMID DNA (52) U.S. Cl...... 424/450; 435/458; 435/320.1; (LIPOGENESIOOd6) AND THERAPEUTIC 514/44; 264/4 AGENTS WITH NUCLEAR LOCALIZATION SIGNAL/FUSOGENIC PEPTIDE CONJUGATES INTO TARGETED LIPOSOME (57) ABSTRACT COMPLEXES (76) Inventor: Teni Boulikas, Mountain View, CA A method is disclosed for encapsulating plasmids, oligo (US) nucleotides or negatively-charged drugs into liposomes hav Correspondence Address: ing a different lipid composition between their inner and Antoinette F. Konski outer membrane bilayers and able to reach primary tumors Baker & McKenzie and their metastases after intravenous injection to animals 660 Hansen Way and humans. The formulation method includes complex formation between DNA with cationic lipid molecules and Palo Alto, CA 94.304 (US) fuSogenic/NLS peptide conjugates composed of a hydro (21) Appl. No.: 09/876,904 phobic chain of about 10-20 amino acids and also containing four or more histidine residues or NLS at their one end. The (22) Filed: Jun. 8, 2001 encapsulated molecules display therapeutic efficacy in eradi cating a variety of Solid human tumors including but not Related U.S. Application Data limited to breast carcinoma and prostate carcinoma. Com bination of the plasmids, oligonucleotides or negatively (60) Provisional application No. 60/210,925, filed on Jun. charged drugs with other anti-neoplastic drugs (the posi 9, 2000. tively-charged cis-platin, doxorubicin) encapsulated into Publication Classification liposomes are of therapeutic value. Also of therapeutic value in cancer eradication are combinations of encapsulated the (51) Int. Cl." ...... A61K 48/00; A61K 9/127; plasmids, oligonucleotides or negatively-charged drugs with C12N 15/88 HSV-tk plus encapsulated ganciclovir. Patent Application Publication Apr. 17, 2003. Sheet 1 of 8 US 2003/0072794 A1

2 Cationic lipids 30%. Ethanol Fusogenics Micelles Peptide

PEG

(MNN Choesteroi

FIGURE Patent Application Publication Apr. 17, 2003 Sheet 2 of 8 US 2003/0072794 A1

FIGURE 2 Patent Application Publication Apr. 17, 2003 Sheet 3 of 8 US 2003/0072794 A1

FIGURE 2 (CONT) Patent Application Publication Apr. 17, 2003 Sheet 4 of 8 US 2003/0072794 A1

FIGURE 3A Patent Application Publication Apr. 17, 2003 Sheet 5 of 8 US 2003/0072794 A1

Patent Application Publication Apr. 17, 2003. Sheet 6 of 8 US 2003/0072794 A1

FIGURE 3C Patent Application Publication Apr. 17, 2003. Sheet 7 of 8 US 2003/0072794 A1

FIGURE 3D Patent Application Publication Apr. 17, 2003. Sheet 8 of 8 US 2003/0072794 A1

S.is x: 3:53.8.388. ; : St. :

is:::::::::: 88....'.

* . . . 3. s

25%

::::: .; :

FIGURE 3E US 2003/0072794 A1 Apr. 17, 2003

ENCAPSULATION OF PLASMD DNA 0006 The primary goals of gene therapy are to repair or (LIPOGENESIOOD6D AND THERAPEUTIC replace mutated genes, regulate gene expression and Signal AGENTS WITH NUCLEAR LOCALIZATION transduction, manipulate the immune System, or target SIGNAL/FUSOGENIC PEPTIDE CONJUGATES malignant and other cells for destruction. See, AnderSon, W. INTO TARGETED LPOSOME COMPLEXES F. (1992) Science 256:808–813; Lasic, D. (1997) in: Lipo somes in Gene Delivery, CRC Press, pp. 1-295; Boulikas, T. CROSS-REFERENCE TO RELATED (1998) Gene Ther. Mol. Biol. 1:1-172; Martin, F. and Bou APPLICATIONS likas, T. (1998) Gene Ther. Mol. Biol. 1:173–214; Ross, G. 0001. This application claims priority under 35 U.S.C. et al. (1996) Hum. Gene Ther. 7:1781-1790. $119(e) to U.S. Provisional Application Serial No. 60/210, 0007 Human cancer presents a particular disease condi 925 filed Jun. 9, 2000. The contents of this application is tion for which effective gene therapy methods would pro hereby incorporated by reference into the present disclosure. vide a particularly useful clinical benefit. Gene therapy concepts for treatment of Such diseases include Stimulation FIELD OF THE INVENTION of immune responses as well as manipulation of a variety of 0002 The present invention relates to the field of gene alternative cellular functions that affect the malignant phe therapy and is Specifically directed toward methods for notype. Although many human tumors are non or weakly producing peptide-lipid-polynucleotide complexes Suitable immunogenic, the immune System can be reinforced and for delivery of polynucleotides to a Subject. The peptide instructed to eliminate cancer cells after transduction of a lipid-polynucleotide complexes. So produced are useful in a patient’s cells ex vivo with the cytokine genes GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-Y, and TNF-C., followed by cell Subject for inhibiting the progression of neoplastic disease. vaccination of the patient (e.g. intradermally) to potentiate T-lymphocyte-mediated antitumor effects (cancer immuno BACKGROUND OF THE INVENTION therapy). DNA vaccination with genes encoding tumor 0003) Throughout this application various publications, antigens and immunotherapy with Synthetic tumor peptide patents and published patent Specifications are referenced by vaccines are further developments that are currently being author and date or by an identifying patent number. Full tested. The genes used for cancer gene therapy in human bibliographical citations for the publications are provided clinical trials include a number of tumor Suppressor genes immediately preceding the claims. The disclosures of these (p53, RB, BRCA1, E1A), antisense oncogenes (antisense publications, patents and published patent specifications are c-fos, c-myc, K-ras), and Suicide genes (HSV-tk, in combi hereby incorporated by reference into the present disclosure nation with ganciclovir, cytosine deaminase in combination to more fully describe the state of the art to which this with 5-fluorocytosine). Other important genes that have invention pertains. been proposed for cancer gene therapy include bcl-2, MDR 1, p21, p16, bax, bcl-XS, E2F, IGF-I, VEGF, angiostatin, 0004 Gene therapy is a newly emerging field of biomedi CFTR, LDL-R, TGF-B, and leptin. One major hurdle pre cal research that holds great promise for the treatment of venting Successful implementation of these gene therapies is both acute and chronic diseases and has the potential to bring the difficulty of efficiently delivering an effective dose of a revolutionary era to molecular medicine. However, despite polynucleotides to the Site of the tumor. Thus, gene delivery numerous preclinical and clinical Studies, routine use of Systems with enhanced transfection capabilities would be gene therapy for the treatment of human disease has not yet highly advantageous. been perfected. It remains an important unmet need of gene therapy to create gene delivery Systems that effectively 0008. A number of different vector technologies and gene target specific cells of interest in a Subject while controlling delivery methods have been proposed and tested for deliv harmful side effects. ering genes in Vivo, including viral vectors and various nucleic acid encapsulation techniques. Alternative viral 0005 Gene therapy is aimed at introducing therapeuti delivery vehicles for genes include murine retroviruses, cally important genes into Somatic cells of patients. Diseases recombinant adenoviral vectors, adeno-associated virus, already shown to be amenable to therapy with gene transfer HSV, EBV, HIV vectors, and baculovirus. Nonviral gene in clinical trials include, cancer (melanoma, breast, lym delivery methods use cationic or neutral liposomes, direct phoma, head and neck, ovarian, colon, prostate, brain, injection of plasmid DNA, and polymers. Various Strategies chronic myelogenous leukemia, non-Small cell lung, lung to enhance efficiency of gene transfer have been tested Such adenocarcinoma, colorectal, neuroblastoma, glioma, glio as fusogenic peptides in combination with liposomes or blastoma, astrocytoma, and others), AIDS, cystic fibrosis, polymers to enhance the release of plasmid DNA from adenosine deaminase deficiency, cardiovascular diseases endoSomes. (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease, C.1-antitrypsin deficiency, rheu 0009. Each of the various gene delivery techniques has matoid arthritis and others. Human diseases expected to be been found to possess different Strengths and weaknesses. the object of clinical trials include hemophilia A and B, Recombinant retroviruses Stably integrate into the chromo Parkinson's disease, ocular diseases, Xeroderma pigmento Some but require host DNA synthesis to insert. Adenoviruses Sum, high blood pressure, obesity. ADA deficiency was the can infect non-dividing cells but cause immune reactions disease Successfully treated by the first human "gene trans leading to the elimination of therapeutically transduced fer” experiment conducted by Kenneth Culver in 1990. See, cells. Adeno-associated virus (AAV) is not pathogenic and Culver, K. W. (1996) in: Gene Therapy: A Primer for does not elicit immune responses but new production Strat Physicians, Second Ed., Mary Ann Liebert, Inc. Publ, New egies are required to obtain high AAV titers for preclinical York, pp. 1-198. and clinical studies. Wild-type AAVs integrate into chromo US 2003/0072794 A1 Apr. 17, 2003

Some 19, whereas recombinant AAVs are deprived of site ing with pre-made lipoSomes or lipids followed by dilution Specific integration and may also persist episomally. in aqueous Solutions and dialysis to remove the ethanol and allow liposome formation and extrusion through membranes 0010 Herpes Simplex Virus (HSV) vectors can infect to a diameter below 160 nm entrapping and encapsulating non-replicating cells, Such as neuronal cells, and has a high DNA with a very high yield. The encapsulated DNA has a payload capacity for foreign DNA but inflict cytotoxic high therapeutic efficacy in eradicating a variety of Solid effects. It seems that each delivery system will be developed human tumors including, but not limited to, breast carci independently of the others and that each will demonstrate noma and prostate carcinoma. A plasmid is constructed with Strengths and weaknesses for certain applications. At DNA carrying anticancer genes including, but not limited to present, retroviruses are most commonly used in human p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, pl6, bax, clinical trials, followed by adenoviruses, cationic liposomes bcl-XS, E2F, IGF-IVEGF, angiostatin, oncostatin, endosta and AAV. tin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-Y, TNF-C., HSV 0.011 AS the challenges of perfecting gene therapy tech tk (in combination with ganciclovir), E. coli cytosine deami niques have become apparent, a variety of additional deliv nase (in combination with 5-fluorocytosine) and is combined ery Systems have been proposed to circumvent the difficul with encapsulated cisplatin or with other Similarly Systemi ties observed with Standard technologies. For example, cally delivered antineoplastic drugs to SuppreSS cancer. cell-based gene delivery using polymer-encapsulated Syn geneic or allogeneic cells implanted into a tissue of a patient BRIEF DESCRIPTION OF THE DRAWINGS can be used to Secrete therapeutic proteins. This method is being tested in trials for amyotrophic lateral Sclerosis using 0014 FIG. 1 illustrates the structure of the cancer tar the ciliary neurotrophic factor gene, and may be extended to geted liposome complex. Factor VIII and IX for hemophilia, interleukin genes, 0.015 FIG. 2 illustrates the results of plasmid DNA dopamine-Secreting cells to treat Parkinson's disease, nerve condensation with various agents as well as various formu growth factor for Alzheimer's disease and other diseases. lation of cationic liposomes in affecting the level of expres Other techniques under development include, vectors with Sion of the reporter beta-galactosidase gene after transfec the Cre-LoxP recombinase system to rid transfected cells of tion of K562 human erythroleukemia cell cultures. undesirable viral DNA sequences, use of tissue-specific promoters to express a gene in a particular cell type, or use 0016 FIG. 3 illustrates tumor targeting in SCID mice. of ligands recognizing cell Surface molecules to direct gene FIG. 3A shows a SCID mouse with a large and small human vehicles to a particular cell type. breast tumor before and after staining with X-Gal to test the expression of the transferred gene. Both tumors turn dark 0012. Additional methods that have been proposed for blue. The intensity of the blue color is proportional to the improving the efficacy of gene therapy technologies include expression of the beta-galactosidase gene. designing p53 "gene bombs' that explode into tumor cells, exploiting the HIV-1 virus to engineer vectors for gene 0017 FIG. 3B shows that in the initial staining of the transfer, combining viruses with polymers or cationic lipids Small tumor, the skin and the intestines at the injection area to improve gene transfer, the attachment of nuclear local are the first organs to turn blue. FIG. 3C is a view of the ization Signal peptides to oligonucleotides to direct genes to back of the animal. The two tumors are clearly visible after nuclei, and the development of molecular Switch Systems removal of the skin (top). Dark Staining of the Small tumor allowing genes to be turned on or off at will. Nevertheless, and light blue Staining of the large tumor is evident at an because of the wide range of disease conditions for which initial stage of staining (bottom). FIG. 3D is a view of the gene therapies are required, and the complexities of devel front side of the animal. The two tumors are clearly visible oping treatments for Such diseases, there remains a need for after removal of the skin. On the figure to the bottom the improved techniques for performing gene therapy. The dark Staining of both tumors is evident at a later Stage during present invention provides methods and compositions for Staining. addressing these issues. 0.018 FIG. 3E shows the front (top) and rear (bottom) higher magnification view of the dark Staining of both DISCLOSURE OF THE INVENTION tumors at a later Stage during Staining. Staining of the 0013 A method is disclosed for encapsulating DNA and vascular System around the Small tumor can also be seen negatively charged drugs into liposomes having a different (bottom). lipid composition between their inner and outer membrane bilayers. The liposomes are able to reach primary tumors BRIEF DESCRIPTION OF THE TABLES and their metastases after intravenous injection to animals and humans. The method includes micelle formation 0019 Table 1 is a list of molecules able to form micelles. between DNA with a mixture of cationic lipid and peptide 0020 Table 2 lists several fusogenic peptides and molecules at molar ratioS to nearly neutralization ratioS in describes their properties, along with a reference. 10-90% ethanol; the cationic peptides specify nuclear local ization and have a hydrophobic moiety endowed with mem 0021 Table 3 lists simple Nuclear Localization Signal brane fusion to improve entrance acroSS the cell membrane (NLS) peptides. of the complex. These peptides insert with their cationic 0022 Table 4 shows a list of “bipartite” or “split” NLS portion directed toward condensed DNA and their hydro peptides. phobic chain buried together with the hydrophobic chains of the lipids in the micelle membrane monolayer. The DNA/ 0023 Table 5 lists “nonpositive NLS' peptides lacking lipid/peptide micelles are converted into liposomes by mix clusters of arginines/lysines. US 2003/0072794 A1 Apr. 17, 2003

0024 Table 6 lists peptides with nucleolar localization 0034. A “gene” refers to a polynucleotide containing at signals (NoLS). least one open reading frame that is capable of encoding a 0.025 Table 7 lists peptides having karyophilic clusters particular polypeptide or protein after being transcribed and on non-membrane protein kinases. translated. 0035 A “gene product” refers to the amino acid (e.g., 0.026 Table 8 lists peptide nuclear localization signals on peptide or polypeptide) generated when a gene is transcribed DNA repair proteins. and translated. 0.027 Table 9 lists NLS peptides in transcription factors. 0036) The following abbreviations are used herein: 0028 Table 10 lists NLS peptides in other nuclear pro DDAB: dimethyldioctadecyl ammonium bromide (same as teins. N,N-distearyl-N,N-dimethylammonium bromide); DODAC: N,N-dioleyl-N,N-dimethylammonium chloride; Modes for Carrying out the Invention DODAP: 1,2-dioleoyl-3-dimethylammonium propane; 0029 Definitions DMRIE: N-1-(2,3-dimyristyloxy)propyl-N,N-dimethyl 0030 The practice of the present invention will employ, N-(2-hydroxyethyl) ammonium bromide; DMTAP: 1,2- unless otherwise indicated, conventional techniques of dimyristoyl-3-trimethylammonium propane; DOGS: Dio immunology, molecular biology, microbiology, cell biology ctadecylamidoglycylspermine; DOTAP (same as DOTMA): and recombinant DNA. These methods are described in the N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium following publications. See, e.g., Sambrook, et al. chloride; DOSPA: N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sper MOLECULAR CLONING: A LABORATORY MANUAL, minecarboxamido)ethyl)-N,N-dimethyl ammonium trifluo d Edition (1989); CURRENT PROTOCOLS IN roacetate; DPTAP: 1,2- dipalmitoyl-3-trimethylammonium MOLECULAR BIOLOGY, F. M. Ausubel, et al. eds., propane; DSTAP: 1,2-disteroyl-3-trimethylammonium pro (1987); the series METHODS IN ENZYMOLOGY (Aca pane; DOPE, 1,2-sn-dioleoylphoshatidylethanolamine; DC demic Press, Inc.); PCR: A PRACTICAL APPROACH, M. Chol, 3B-(N-(N',N'-dimethylaminoethane)carbamoyl MacPherson, et al., IRL Press at Oxford University Press )cholesterol. See, Gao et al., Biochem. BiophyS. Res. Comm. (1991); PCR 2: A PRACTICAL APPROACH, MacPherson 179:280-285 (1991). et al., eds. (1995); ANTIBODIES, A LABORATORY 0037 AS used herein, the term “pharmaceutically accept MANUAL, Harlow and Lane, eds. (1988); and ANIMAL able anion” refers to anions of organic and inorganic acids CELL CULTURE, R. I. Freshney, ed. (1987). that provide non-toxic Salts in pharmaceutical preparations. 0.031 AS used in the specification and claims, the Singu Examples of Such anions include the halides anions, chlo lar form “a,”“an” and “the” include plural references unless ride, bromide, and iodide, inorganic anions Such as Sulfate, the context clearly dictates otherwise. For example, the term phosphate, and nitrate, and organic anions. Organic anions “a cell' includes a plurality of cells, including mixtures may be derived from Simple organic acids, Such as acetic thereof. acid, propionic acid, glycolic acid, pyruvic acid, Oxalic acid, malic acid, malonic acid, Succinic acid, maleic, acid, fumaric 0.032 The term “comprising” is intended to mean that the acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, compositions and methods include the recited elements, but mandelic acid, methane Sulfonic acid, ethane Sulfonic acid, not excluding others. “Consisting essentially of when used p-tolueneSulfonic acid, and the like. The preparation of to define compositions and methods, Shall mean excluding pharmaceutically acceptable Salts is described in Berge, et other elements of any essential Significance to the combi al., J. Pharm. Sci. 66:1-19 (1977), incorporated herein by nation. Thus, a composition consisting essentially of the reference. elements as defined herein would not exclude trace contami nants from the isolation and purification method and phar 0038 Physiologically acceptable carriers, excipients or maceutically acceptable carriers, Such as phosphate buffered Stabilizers are nontoxic to recipients at the dosages and Saline, preservatives, and the like. “Consisting of shall concentrations employed, and include bufferS Such as phos mean excluding more than trace elements of other ingredi phate, citrate, and other organic acids, antioxidants includ ents and Substantial method Steps for administering the ing ascorbic acid; low molecular weight (less than about 10 compositions of this invention. Embodiments defined by residues) polypeptides, proteins, Such as Serum albumin, each of these transition terms are within the Scope of this gelatin, or immunoglobulins, hydrophilic polymerS Such as invention. polyvinylpyrrollidone; amino acids Such as glycine, 0033. The terms “polynucleotide' and “nucleic acid mol glutamine, asparagine, arginine or lysine; monosaccharides, ecule' are used interchangeably to refer to polymeric forms disaccharides, and other carbohydrates including glucose, of nucleotides of any length. The polynucleotides may mannose, or dextrins; chelating agents Such as EDTA: Sugar contain deoxyribonucleotides, ribonucleotides, and/or their alcohols Such as mannitol or Sorbitol, Salt-forming counter analogs. Nucleotides may have any three-dimensional Struc ions Such as Sodium; and/or nonionic Surfactants Such as ture, and may perform any function, known or unknown. Tween, Pluronics or polyethylene glycol (PEG). PEG mol The term “polynucleotide' includes, for example, Single-, ecules also contain a fusogenic peptide with an attached double-Stranded and triple helical molecules, a gene or gene Nuclear Localization Signal (NLS) covalently linked to the fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, end of the PEG molecule. cDNA, recombinant polynucleotides, branched polynucle 0039. The term “cationic lipid” refers to any of a number otides, plasmids, vectors, isolated DNA of any Sequence, of lipid Species that carry a net positive charge at physi isolated RNA of any Sequence, nucleic acid probes, and ological pH. Such lipids include, but are not limited to, primers. A nucleic acid molecule may also comprise modi DDAB, DMRIE, DODAC, DOGS, DOTAP, DOSPA and fied nucleic acid molecules. DC-Chol. Additionally, a number of commercial prepara US 2003/0072794 A1 Apr. 17, 2003

tions of cationic lipids are available that can be used in the amount of a fusogenic/NLS peptide conjugate. Examples of present invention. These include, for example, LIPOFEC fuSogenic/NLS peptide conjugates include, but are not lim TIN (commercially available cationic liposomes comprising ited to (KAWLKAF) (SEQ ID NO:1), GLFKAAAKLLK DOTMA and DOPE, from GIBCO/BRL, Grand Island, SLWKLLLKA (SEQ ID NO:2), LLLKAFAKLLKSL N.Y., USA); LIPOFECTAMINE (commercially available WKLLLKA (SEQID NO:3), as well as all derivatives of the cationic liposomes comprising DOSPA and DOPE, from prototype (Hydrophobic3-Karyophilic1-Hydrophobic2 GIBCO/BRL); and TRANSFECTAM (commercially avail Karyophilic1), where Hydrophobic is any of the A, I, L, V, able cationic lipids comprising DOGS in ethanol from P. G., W, F and Karyophilic is any of the K, R, or H, Promega Corp., Madison, Wis., USA). containing a positively-charged residue every 3rd or 4th amino acid, which form alpha helices and direct a net 0040. This invention further provides a number of meth positive charge to the same direction of the helix. Additional ods for producing micelles with entrapped therapeutic drugs. examples include but are not limited to GLFKAIAGFIKNG The method is particularly useful to produce micelles of WKGMIDGGGYC (SEQ ID NO:4) from influenza virus drugs or compositions having a net overall negative charge, hemagglutinin HA-2; YGRKKRRQRRR (SEQ ID NO:5) e.g., DNA, RNA or negatively charged Small molecules. For from TAT of HIV; MSGTFGGILAGLIGLL(K/R/H), example, the DNA can be comprised within a plasmid vector (SEQ ID NO:6), derived from the N-terminal region of the and encode for a therapeutic protein, e.g., wild-type p53, S protein of duck hepatitis B virus, but with the addition of HSV-tk, p21, Bax, Bad, IL-2, IL-12, GM-CSF, angiostatin, one to Six positively-charged lysine, arginine or histidine endostatin and oncostatin. In one embodiment, the method residues, and combinations of these, able to interact directly requires combining an effective amount of the therapeutic with the phosphate groups of plasmid or oligonucleotide agent with an effective amount of cationic lipids. Cationic DNA, compensating for part of the positive charges pro lipids useful in the methods of this invention include, but are vided by the cationic lipids. GAAIGLAWIPYFGPAA (SEQ not limited to, DDAB, dimethyldioctadecyl ammonium bro ID NO:7) is derived from the fusogenic peptide of the Ebola mide; DMRIE: N-1-(2,3-dimyristyloxy)propyl-N,N-dim virus transmembrane protein; residues 53-70 (C-terminal ethyl-N-(2-hydroxyethyl) ammonium bromide; DMTAP: helix) of apolipoprotein (apo) AII peptide; the 23-residue 1,2-dimyristoyl-3-trimethylammonium propane; DOGS: fusogenic N-terminal peptide of HIV-1 transmembrane gly Dioctadecylamidoglycylspermine; DOTAP (same as coprotein gp41, the 29-42-residue fragment from Alzhe DOTMA): N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethy imer's B-amyloid peptide; the fusion peptide and N-terminal lammonium chloride; DPTAP: 1,2-dipalmitoyl-3-trimethy heptad repeat of Sendai virus; the 56-68 helical segment of lammonium propane; DSTAP: 1,2-disteroyl-3-trimethylam lecithin cholesterol acyltransferase. Included within these monium propane. embodiments are shorter versions of these peptides, that are 0041. In one aspect, a ratio of from about 30 to about 90% known to induce fusion of unilamellar lipid vesicles or all of phosphates contained within the negatively charged thera that are similarly derivatized with the addition of one to six peutic agent are neutralized by positive charges on lipid positively-charged lysine, arginine or histidine residues molecules (negative charges are in excess) to form an (K/R/H), able to interact directly with the phosphate groups electroStatic micelle complex in an effective concentration of plasmid or oligonucleotide DNA, compensating for part of ethanol. In one aspect, the ethanol Solution is from about of the positive charges provided by the cationic lipids. The 20% to about 80% ethanol. In a further aspect, the ethanol fuSogenic peptides in the fusogenic/NLS conjugates repre concentration is about 30%. The ethanol/cationic lipid/ Sent hydrophobic amino acid stretches, and Smaller frag therapeutic agent complex is then combined with an effec ments of these peptide Sequences, that include all signal tive amount of a fusogenic-karyophilic peptide conjugate. In peptide Sequences used in membrane or Secreted proteins one aspect, an effective amount of the conjugate is a ratio that insert into the endoplasmic reticulum. Alternatively, the range from about 0.0 to about 0.3 (positive charges on conjugates represent transmembrane domains and Smaller peptide to negative charges on phosphate groups) to neu fragments of these peptide Sequences. tralize the majority of the remaining negative charges on the 0044) In one aspect of the invention, the NLS peptide phosphate groups of the therapeutic agents thereby leading component in fusogenic/NLS peptide conjugates is derived to an almost complete neutralization of the complex. The from the fusogenic hydrophobic peptides. However, there is optimal conditions give to the complex a slightly negative an addition of 5-6 amino acid karyophilic Nuclear Local charge. However, when the positive charges on cationic ization Signals (NLS) derived from a number of known NLS lipids exceed the negative charges on the DNA, the exceSS peptides, as well as from Searches of the nuclear protein of positive charges are neutralized by DPPG (dipalmitoyl databases, for Stretches of five or more karyophilic amino phosphatidyl glycerol) and its derivatives, or by other acid stretches in proteins containing at least four positively anionic lipid molecules in the final micelle complex. charged amino aids flanked by a proline (P) or glycine (G). Examples of NLS peptides are shown in Tables 1-8. The 0042. In an alternative embodiment, the above methods NLS peptide component in fusogenic/NLS peptide conju can be modified by addition of DNA condensing agents gates are Synthetic peptides containing the above Said NLS, Selected from Spermine, Spermidine, and magnesium or but further modified by additional K, R, H residues at the other divalent metal ions neutralizing a certain percentage central part of the peptide or with P or G at the N- or (1-20%) of phosphate groups. C-terminus. 0043. In a further embodiment, the cationic lipids are 0045. In a further aspect, the fusogenic/NLS peptide combined with an effective amount of fusogenic lipid DOPE conjugates are derived from the Said fusogenic hydrophobic at various molar ratioS for example, in a molar ratio of from peptides but with the addition of a stretch of Ho (four to six about 1:1 cationic lipid:DOPE. In an alternative embodi histidine residues) in the place of NLS. Micelle formation ment, the cationic lipids are combined with an effective takes place at pH 5-6 where histidyl residues are positively US 2003/0072794 A1 Apr. 17, 2003

charged but lose their charge at the nearly neutral pH of the liver, Spleen and bone marrow tissue, to produce a Secreted biological fluids, thus releasing the plasmid or oligonucle form of a therapeutic protein. otide DNA from their electrostatic interaction. 0052 The disclosed therapies also provide methods for 0046) The fusogenic peptide/NLS peptide conjugates are reducing tumor Size by combining the encapsulated plasmid linked to each other with a short amino acid Stretch repre DNA carrying one or more anticancer genes Selected from Senting an endogenous protease cleavage Site. the group consisting of p53, RB, BRCA1, E1A, bc1-2, MDR-1, p21, p16, bax, bc1-XS, E2F, IGF-IVEGF, angiosta 0047. In a preferred aspect of the invention, the structure tin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, of the preferred prototype fusogenic/NLS peptide conjugate IFN-Y, TNF-C, HSV-tk (in combination with ganciclovir), E. used in this invention is: PKKRRGPSP(L/A/I) (SEQ ID coli cytosine deaminase (in combination with 5-fluorocy NO:8), where (L/A/I) is a stretch of 12-20 hydrophobic tosine) with encapsulated antisense oligonucleotides (anti amino acids containing A, L., I, Y, W, F and other hydro Sense c-fos, c-myc, K-ras), ribozymes or triplex-forming phobic amino acids. oligonucleotides directed against genes that control the cell 0.048. The micelles made by the above methods are cycle or signaling pathways. These methods can be modified further provided by this invention by conversion into lipo by combining the encapsulated plasmid DNA carrying one Somes. An effective amount of liposomes (diameter from or more anticancer genes of with encapsulated or free about 80 to about 160 nm), or of a lipid solution composed antineoplastic drugs, consisting of the group of adriamycin, of cholesterol (from about 10% to about 50%), neutral angiostatin, azathioprine, bleomycin, buSulfane, camptoth phospholipid Such as hydrogenated Soy phosphatidylcholine ecin, carboplatin, carmustine, chlorambucile, chlo (HSPC) (from about 40% to about 90%), and the derivatized rmethamine, chloroquinoxaline Sulfonamide, cisplatin, vesicle-forming lipid PEG-DSPE (distearoylphosphatidyl cyclophosphamide, cycloplatam, cytarabine, dacarbazine, ethanolamine) from about 1-to about 7 mole percent, is dactinomycin, daunorubicin, didox, doxorubicin, endostatin, added to the micelle Solution. enloplatin, estramustine, etoposide, extramustinephosphat, flucytosine, fluorodeoxyuridine, fluorouracil, gallium 0049. In a specific embodiment, the liposomes are com nitrate, hydroxyurea, idoxuridine, interferons, interleukins, posed of Vesicle-forming lipids and between from about 1 to leuprolide, lobaplatin, lomustine, mannomustine, mechlore about 7 mole percent of distearoylphosphatidyl ethanola thamine, mechlorethaminoxide, melphalan, mercaptopurine, mine (DSPE) derivatized with a polyethyleneglycol. The methotrexate, mithramycin, mitobronitole, mitomycin, composition of claim 20, wherein the polyethyleneglycol mycophenolic acid, nocodazole, oncostatin, Oxaliplatin, has a molecular weight is between about 1,000 to 5,000 paclitaxel, pentamustine, platinum-triamine compleX, plica daltons. Micelles are converted into liposomes with a con mycin, prednisolone, prednisone, procarbazine, protein comitant decrease of the ethanol concentration which can be kinase C inhibitors, puromycine, Semustine, Signal transduc accomplished by removal of the ethanol by dialysis of the tion inhibitors, Spiroplatin, Streptozotocine, Stromelysin lipoSome complexes through permeable membranes or inhibitors, taxol, tegafur, telomerase inhibitors, teniposide, reduced to a diameter of 80-160 nm by extrusion through thalidomide, thiamiprine, thioguanine, thiotepa, tiamiprine, membranes. tretamine, triaziquone, trifosfamide, tyrosine kinase inhibi 0050 Liposome encapsulated therapeutic agents pro tors, uramustine, Vidarabine, vinblastine, Vinca alcaloids, duced by the above methods are further provided by this Vincristine, Vindesline, Vorozole, Zeniplatin, Zeniplatin, and invention. Zinostatin. 0051. Also provided herein is a method for delivering a 0053. The following examples are intended to illustrate, therapeutic agent Such as plasmid DNA or oligonucleotides but not limit the invention. to a tissue cell in Vivo by intravenous, or other type of 0054 Liposome Composition injection of the micelles or liposomes. This method specifi cally targets a primary tumor and the metastases by the long 0055 Liposomes are microscopic vesicles consisting of circulating time of the micelle or liposome complex because concentric lipid bilayers. Structurally, liposomes range in of the exposure of PEG chains on its surface, its small size Size and shape from long tubes to Spheres, with dimensions (80-160 nm) and the decrease in hydrostatic pressure in the from a few hundred Angstroms to fractions of a millimeter. Solid tumor from the center to its periphery Supporting a Vesicle-forming lipids are Selected to achieve a Specified preferential extravasation through the tumor vasculature to degree of fluidity or rigidity of the final complex providing the extracellular space in tumors. A method for delivering the lipid composition of the outer layer. These are neutral plasmid or oligonucleotide DNA acroSS the cell membrane (cholesterol) or bipolar and include phospholipids, Such as barrier of the tumors using the micelle or lipoSome com phosphatidylcholine (PC), phosphatidylethanolamine (PE), plexes described herein is capable because of the presence of phosphatidylinositol (PI), and sphingomyelin (SM) and the fusogenic peptides in the complex. In particular, a other type of bipolar lipids including but not limited to method for delivering plasmid or oligonucleotide DNA to dioleoylphosphatidylethanolamine (DOPE), with a hydro the liver, Spleen and bone marrow after intravenous injection carbon chain length in the range of 14-22, and Saturated or of the complexes is provided. Further provided is a method with one or more double C=C bonds. Examples of lipids for delivering therapeutic genes to the liver, Spleen and bone capable of producing a stable liposome, alone, or in com marrow of cancer and noncancer patients including but not bination with other lipid components are phospholipids, limited to, factor VIII or IX for the therapy of hemophilias, such as hydrogenated soy phosphatidylcholine (HSPC), multidrug resistance, cytokine genes for cancer immuno lecithin, phosphatidylethanolamine, lySolecithin, lySophos therapy, genes for the alleviation of pain, genes for the phatidylethanolamine, phosphatidylserine, phosphatidyli alleviation of diabetes and genes that can be introduced to nositol, Sphingomyelin, cephalin, cardiolipin, phosphatidic US 2003/0072794 A1 Apr. 17, 2003 acid, cerebrosides, distearoylphosphatidylethanolamine 0060 Preparing Cationic Liposome/Cationic Peptide/ (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmi Nucleic Acid Micelles toylphosphatidylcholine (DPPC), palmitoyloleoylphos phatidylcholine (POPC), palmitoyloleoylphosphatidyle 0061 Cationic lipids, with the exception of sphingosine thanolamine (POPE) and dioleoylphosphatidylethanolamine and Some lipids in primitive life forms, do not occur in 4-(N-maleimido-methyl)cyclohexane-1-carboxylate nature. The present invention uses Single-chain amphiphiles (DOPE-mal). Additional non-phosphorous containing lipids which are chloride and bromide salts of the alkyltrimethy that can become incorporated into liposomes include Steary lammonium Surfactants including but not limited to C12 and lamine, dodecylamine, hexadecylamine, isopropyl C16 chains abbreviated DDAB (same as DODAB) or myristate, triethanolamine-lauryl Sulfate, alkyl-aryl Sulfate, CTAB. The molecular geometry of these molecules deter acetyl palmitate, glycerol ricinoleate, hexadecyl Stereate, mines the critical micelle concentration (ratio between free amphoteric acrylic polymers, polyethyloxylated fatty acid monomers in Solution and molecules in micelles). Lipid amides, and the cationic lipids mentioned above (DDAB, eXchange between the two States is a highly dynamic pro DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), ceSS, phospholipids have critical micelle concentration Val DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged ues below 10 M and are more stable in liposomes; how lipids include phosphatidic acid (PA), dipalmitoylphosphati ever, Single chain detergents, Such as Stearylamine, may dylglycerol (DPPG), dioleoylphosphatidylglycerol and emerge from the liposome membrane upon dilution or (DOPG), dicetylphosphate that are able to form vesicles. intravenous injection in milliseconds (Lasic, 1997). Preferred lipids for use in the present invention are choles 0062 Cationic lipids include, but are not limited to, terol, hydrogenated soy phosphatidylcholine (HSPC) and, DDAB: dimethyldioctadecyl ammonium bromide (same as the derivatized vesicle-forming lipid PEG-DSPE. N,N-distearyl-N,N-dimethylammonium bromide); DMRIE: 0056 Typically, liposomes can be divided into three N-1-(2,3-dimyristyloxy)propyl-N,N-dimethyl-N-(2-hy categories based on their overall size and the nature of the droxyethyl) ammonium bromide; DODAC: N,N-dioleyl-N, lamellar Structure. The three classifications, as developed by N-dimethylammonium chloride; DMTAP: 1,2-dimyristoyl the New York Academy Sciences Meeting, “Liposomes and 3-trimethylammonium propane; DODAP: 1,2-dioleoyl-3- dimethylammonium propane, DOGS: Their Use in Biology and Medicine,” December 1977, are Dioctadecylamidoglycylspermine; DOTAP (same as multi-lamellar vesicles (MLVs), small uni-lamellar vesicles DOTMA): N-(1-(2,3-dioleoyloxy)propyl)-N.N.N-trimethy (SUVs) and large uni-lamellar vesicles (LUVs). lammonium chloride; DOSPA: N-(1-(2,3-dioleyloxy)pro 0057 SUVs range in diameter from approximately 20 to pyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl 50 nm and consist of a Single lipid bilayer Surrounding an ammonium trifluoroacetate; DPTAP: 1,2-dipalmitoyl-3-tri aqueous compartment. Unilamellar vesicles can also be methylammonium propane; DSTAP: 1,2-disteroyl-3-trim prepared in sizes from about 50 nm to 600 nm in diameter. ethylammonium propane; DC-Chol, 3 B-(N-(N',N'-dim While unilamellar are Single compartmental vesicles of ethylaminoethane)carbamoyl)cholesterol. fairly uniform size, MLVs vary greatly in size up to 10,000 nm, or thereabouts, are multi-compartmental in their struc 0063 Lipid-based vectors used in gene transfer have ture and contain more than one bilayer. LUV liposomes are been formulated in one of two ways. In one method, the So named because of their large diameter that ranges from nucleic acid is introduced into preformed liposomes made of about 600 nm to 30,000 nm, they can contain more than one mixtures of cationic lipids and neutral lipids. The complexes bilayer. thus formed have undefined and complicated Structures and the transfection efficiency is Severely reduced by the pres 0.058 Liposomes may be prepared by a number of meth ence of Serum. Preformed liposomes are commercially ods not all of which produce the three different types of available as LIPOFECTIN and LIPOFECTAMINE. The liposomes. For example, ultraSonic dispersion by means of second method involves the formation of DNA complexes immersing a metal probe directly into a Suspension of MLVS with mono- or poly-cationic lipids without the presence of a is a common way for preparing SUVs. neutral lipid. These complexes are prepared in the presence of ethanol and are not stable in water. Additionally, these 0059 Preparing liposomes of the MLV class usually complexes are adversely affected by Serum (see, Behr, Acc. involves dissolving the lipids in an appropriate organic Chem. Res. 26:274-78 (1993)). An example of a commer Solvent and then removing the Solvent under a gas or air cially available poly-cationic lipid is TRANSFECTAM. stream. This leaves behind a thin film of dry lipid on the Other efforts to encapsulate DNA in lipid-based formula Surface of the container. An aqueous Solution is then intro tions have not overcome these problems (see, Szoka et al., duced into the container with Shaking, in order to free lipid Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer, U.S. material from the Sides of the container. This process dis perses the lipid, causing it to form into lipid aggregates or Pat. No. 4,515,736). liposomes. Liposomes of the LUV variety may be made by 0064. The nucleotide polymers can be single-stranded slow hydration of a thin layer of lipid with distilled water or DNA or RNA, or double-stranded DNA or DNA-RNA an aqueous Solution of Some Sort. Alternatively, liposomes hybrids. Examples of double-stranded DNA include struc may be prepared by lyophilization. This process comprises tural genes, genes including control and termination regions, drying a Solution of lipids to a film under a stream of and Self-replicating Systems. Such as plasmid DNA. Particu nitrogen. This film is then dissolved in a volatile solvent, larly preferred nucleic acids are plasmids. Single-Stranded frozen, and placed on a lyophilization apparatus to remove nucleic acids include antisense oligonucleotides (comple the Solvent. To prepare a pharmaceutical formulation con mentary to DNA and RNA), ribozymes and triplex-forming taining a drug, a Solution of the drug is added to the oligonucleotides. In order to increase Stability, Some single lyophilized lipids, whereupon liposomes are formed. stranded nucleic acids will preferably have some or all of the US 2003/0072794 A1 Apr. 17, 2003 nucleotide linkages Substituted with Stable, non-phosphodi may be prepared by lyophilization. This proceSS comprises ester linkages, including, for example, phosphorothioate, drying a Solution of lipids to a film under a stream of phosphorodithioate, phosphoroSelenate, methylphospho nitrogen. The film is then dissolved in a volatile solvent, nate, or O-alkyl phosphotriester linkages. frozen, and placed on a lyophilization apparatus to remove the Solvent. To prepare a pharmaceutical formulation con 0065 Encapsulating Cationic Liposome/Cationic Pep taining a drug, a Solution of the drug is added to the tide/Nucleic Acid Micelles into Neutral Liposomes lyophilized lipids, whereupon liposomes are formed. 0.066 Cationic lipids used with fusogenic peptide/NLS 0071. Following liposome preparation, the liposomes conjugates to provide the inner layer of the particle can be may be sized to achieve a desired size range and relatively any of a number of Substances Selected from the group of narrow distribution of liposome sizes. Preferably, the pre DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP formed liposomes are sized to a mean diameter of about 80 (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol. The cat to 160 nm (the upper size limit for filter sterilization before ionic lipid is combined with DOPE. In one group of embodi in Vivo administration). Several techniques are available for ments, the preferred cationic lipid is DDAB:DOPE 1: 1. sizing liposomes to a desired size. Sonicating a liposome 0067 Neutral lipids used herein to provide the outer layer Suspension either by bath or probe Sonication produces a of the particles can be any of a number of lipid Species that progressive size reduction down to Small unilamellar exist either in an uncharged or neutral Zwitterionic form at vesicles less than about 0.05 microns (50 nm) in size. physiological pH. Such lipids are Selected from a group Extrusion of lipoSome through a Small-pore polycarbonate is consisting of diacylphosphatidylcholine, diacylphosphati our preferred method for reducing lipoSome sizes to a dylethanolamine, ceramide, Sphingomyelin, cephalin, and relatively well-defined size distribution. The liposomes may cerebrosides. In one group of embodiments, lipids contain be extruded through Successively Smaller-pore membranes, ing Saturated, mono-, or di-unsaturated fatty acids with to achieve a gradual reduction in liposome size. carbon chain lengths in the range of C14 to C22 are 0072) One way used to coat DNA with lipid is by preferred. In general, leSS Saturated lipids are more easily controlled detergent depletion from a cationic lipid/DNA/ sized, particularly when the liposomes must be sized below detergent complex. This method can give complexes with about 0.16 microns, for purposes of filter Sterilization. stability in plasma. Hofland et al. (1996), have prepared such Consideration of liposome size, rigidity and Stability of the complexes by dialysis of a mixture of DOSPA/DOPE/DNA/ liposomes in the final preparation, its shelf life without octylglucoside. leakage of the encapsulated DNA, and Stability in the bloodstream generally guide the selection of neutral lipids 0.073 Pharmaceutical compositions comprising the cat for providing the outer coating of our gene vehicles. Lipids ionic lipoSome/nucleic acid complexes of the invention are having a variety of acyl chain groups of varying chain length prepared according to Standard techniques and further com and degree of Saturation are available or may be isolated or prise a pharmaceutically acceptable carrier. Generally, nor Synthesized by well-known techniques. In another group of mal Saline will be employed as the pharmaceutically accept embodiments, lipids with carbon chain lengths in the range able carrier. of C14 to C22 are used. Preferably, the neutral lipids used in 0074 For in vivo administration, the pharmaceutical the present invention are hydrogenated Soy phosphatidyl compositions are preferably administered parenterally, i.e., choline (HSPC), cholesterol, and PEG-distearoylphosphati intravenously, intraperitoneally, Subcutaneously, intrathe dyl ethanolamine (DSPE) or PEG-ceramide. cally, injection to the Spinal cord, intramuscularly, intraar 0068 Methods for Preparing Liposomes ticularly, portal vein injection, or intratumorally. More pref erably, the pharmaceutical compositions are administered 0069. A variety of methods for preparing various lipo intravenously or intratumorally by a bolus injection. In other Some forms have been described in Several issued patents, methods, the pharmaceutical preparations may be contacted for example, U.S. Pat. Nos. 4,229,360; 4,224,179; 4,241, with the target tissue by direct application of the preparation 046; 4,737,323; 4,078,052; 4,235,871; 4,501,728; and to the tissue. The application may be made by topical “open’ 4,837,028, as well as in the articles Szoka et al., Ann. Rev. or “closed” procedures. The term “topical” means the direct Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. application of the pharmaceutical preparation to a tissue Lip. 40:89 (1986). These methods do not produce all three different types of liposomes (MLVs, SUVs, LUVs). For exposed to the environment, Such as the skin, to any Surface example, ultraSonic dispersion by means of immersing a of the body, nasopharynx, external auditory canal, ocular metal probe directly into a Suspension of MLVs is a common administration and administration to the Surface of any body way for preparing SUVs. cavities, inhalation to the lung, genital mucosa and the like. 0075 “Open” procedures are those procedures that 0070 Preparing liposomes of the MLV class usually include incising the skin of a patient and directly visualizing involves dissolving the lipids in an appropriate organic the underlying tissue to which the pharmaceutical prepara Solvent and then removing the Solvent under a gas or air tions are applied. This is generally accomplished by a stream. This leaves behind a thin film of dry lipid on the Surgical procedure, Such as a thoracotomy to access the Surface of the container. An aqueous Solution is then intro lungs, abdominal laparotomy to access abdominal Viscera, duced into the container with Shaking, in order to free lipid or other direct Surgical approach to the target tissue. material from the Sides of the container. This process dis perses the lipid, causing it to form into lipid aggregates or 0076 “Closed” procedures are invasive procedures in liposomes. Liposomes of the LUV variety may be made by which the internal target tissues are not directly visualized, slow hydration of a thin layer of lipid with distilled water or but accessed via insertion of instruments through Small an aqueous Solution of Some Sort. Alternatively, liposomes wounds in the skin. For example, the preparations may be US 2003/0072794 A1 Apr. 17, 2003 administered to the peritoneum by needle lavage. Likewise, when non-dividing cells are targeted, Such as liver, Spleen or the pharmaceutical preparations may be administered to the bone marrow cells that represent the major sites for extrava meninges or Spinal cord by infusion during a lumbar punc sation and concentration of these vehicles other than Solid ture followed by appropriate positioning of the patient as tumorS. commonly practiced for Spinal anesthesia or metraZamide imaging of the Spinal cord. Alternatively, the preparations 0085 Organic Solvent may be administered through endoscopic devices. 0086 A suitable solvent for preparing a micelle from the desired lipid components is ethanol, methanol, or other EXAMPLES aliphatic alcohols Such as propanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol. Mixtures of 0077. Materials and Methods two or more Solvents may be used in the practice of the 0078 DDAB, DOPE (dioleoylphosphatidylethanola invention. It is also to be understood that any Solvent that is mine) and most other lipids used here were purchased from miscible with an ethanol Solution, even in Small amounts, Avanti Polar Lipids; PEG-DSPE was from Syngena. can be used to improve micelle formation and its Subsequent conversion into liposomes, including chloroform, dichlo 0079 Engineering of Plasmid pLF romethane, diethylether, cyclohexane, cyclopentane, ben 0080) The pGL3-C (Promega) was cut with Xbal and Zene, and toluene. blunt-end ligated using the Klenow fragment of E. coli DNA polymerase. It was then cut with HindIII and the 1689-bp 0087 Cationic Lipids fragment, carrying the luciferase gene, was gel-purified. The 0088. In a further embodiment, the liposome encapsu pGFP-N1 plasmid (Clontech) was cut with SmaI and Hin lated DNA described herein further comprises an effective dIII and the 4.7 kb fragment, isolated from an agarose gel, amount of cationic lipids. Cationic lipids have been widely was ligated with the luciferase fragment. JM109 E. Coli used for gene transfer; a number of clinical trials (34 out of cells were transformed and 20 colonies were Selected; about 220 total RAC-approved protocols as of December, 1997) half of them showed the presence of inserts; 8 clones with use cationic lipids. Although many cell culture Studies have inserts were cut with BamHI and XhoI to further confirm the been documented, Systemic delivery of genes with cationic presence of the luciferase gene, Seven of them were positive. lipids in vivo has been very limited. All clinical protocols use Subcutaneous, intradermal, intratumoral, and intracra 0.081 Radiolabeled plasmid pLF was generated by cul nial injection as well as intranasal, intrapleural, or aeroSol turing Escherichia coli in H-thymidine-5'-triphosphate or administration but not I.V. delivery, because of the toxicity Pinorganic phosphate (5 mCi) (Dupont/NEN, Boston, of the cationic lipids and DOPE (see, Martin and Boulikas, Mass.) and purified using standard techniques as described 1998). Liposomes formulated from DOPE and cationic above. lipids based on diacyltrimethylammonium propane (dio 0082) DLS Measurements leoyl-, dimyristoyl-, dipalmitoyl-, disteroyl-trimethylammo nium propane or DOTAP, DMTAP, DPTAP, DSTAP, respec 0083) A Coulter N4M light scattering instrument was tively) or DDAB were highly toxic when incubated in vitro used, at a 90° angle, set at a run time of 200 sec, using 4 to with phagocytic cells (macrophages and U937 cells), but not 25 microSec Sample time. The Scan of the particle size towards non-phagocytic T lymphocytes. The rank order of distribution was obtained in 1 ml Sample Volume using toxicity was DOPE/DDAB >DOPE/DOTAP >DOPE/ plastic cuvettes, at 20° C. and at 0.01 poise viscosity. DMTAP >DOPE/DPTAP >DOPE/DSTAP; and the toxicity 0084. In one aspect, this invention provides a method for was determined from the effect of the cationic liposomes on entrapping DNA into lipids that enhances the content of the synthesis of nitric oxide (NO) and TNF-C. produced by plasmid per Volume unit, and reduces the toxicity of the activated macrophages (Filion and Phillips, 1997). cationic lipids used to trap plasmid or oligonucleotide DNA. 0089 Another aspect to be considered before I.V. injec The DNA becomes hidden in the inner membrane bilayer of tion is undertaken, is that negatively charged Serum proteins the final complex. Furthermore, the gene transfer complex is can interact and cause inactivation of cationic liposomes endowed with long circulation time in body fluids and (Yang and Huang, 1997). Condensing agents used for plas extravasateS preferentially into Solid tumors and their meta mid delivery including polylysine, transferrin-polylysine, a Static foci and nodules. The extravasation occurs through fifth-generation poly(amidoamine) (PAMAM) dendrimer, their vasculature at most sites of the human or animal body poly(ethyleneimine), and several cationic lipids (DOTAP, after intravenous injection of the gene-carrying vehicles. DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE), were This occurs because of their small size (100-160 nm), their found to activate the complement System to varying extents. content in neutral to slightly negatively-charged lipids in Strong complement activation was seen with long-chain their outer membrane bilayers, and their coating with PEG. polylysines, the dendrimer, poly(ethyleneimine), and These gene delivery vehicles are able to cross the cell DOGS. Modifying the Surface of preformed DNA com membrane barrier after they reach the extracellular tumor Space because of the presence of fusogenic peptides conju plexes with polyethyleneglycol (Plank et al., 1996) consid gated with karyophilic peptides. The vehicles assume a erably reduced complement activation. certain predefined orientation in the lipid membrane with 0090 Cationic lipids increase the transfection efficiency their positive ends directed toward DNA and their hydro by destabilizing the biological membranes, including phobic tail buried inside the hydrophobic lipid bilayer. The plasma, endoSomal, and lySOSomal membranes. Incubation labile NLS-fuSogenic peptide linkage is cleaved after of isolated lysosomes with low concentrations of DOTAP endocytosis and the remaining NLS peptide bound to plas caused a Striking increase in free activity of B-galactosidase, mid DNA aids its nuclear uptake. This occurs especially and even a release of the enzyme into the medium. This US 2003/0072794 A1 Apr. 17, 2003 demonstrates that the lySOSomal membrane is deeply desta 0096 Plasmid Condensation With Spermine bilized by the lipid. The mechanism of destabilization was 0097. In a further embodiment, the liposome encapsu thought to involve an interaction between cationic liposomes lated DNA described herein is condensed with spermine and anionic lipids of the lySOSomal membrane, thus allowing and/or Spermidine. DNA can be presented to cells in culture a fusion between the lipid bilayers. The proceSS was leSS as a complex with polycations Such as polylysine, or basic pronounced at pH 5 than at pH 7.4, and anionic amphipathic proteins Such as protamine, total histones or Specific histone lipids were able to prevent partially this membrane desta fractions, protamine (Boulikas and Martin, 1997). The inter bilization (Wattiaux et al., 1997). action of plasmid DNA with protamine sulfate, followed by the addition of DOTAP cationic liposomes, offered a better 0091. In contrast to DOTAP and DMRIE that were 100% protection of plasmid DNA against enzymatic digestion. The charged at pH 7.4, DC-CHOL was only about 50% charged method gave consistently higher gene expression in mice via as monitored by a pH-sensitive fluorophore. This difference tail vein injection as compared with DOTAP/DNA com decreases the charge on the external Surfaces of the lipo plexes. 50 lug of luciferase-plasmid per mouse gave 20 ng Somes, and was proposed to promote an easier dissociation luciferase protein per mg extracted tissue protein in the lung, of bilayers containing DC-CHOL from the plasmid DNA, that was detected as early as 1 h after injection, peaked at 6 and an increase in release of the DNA-lipid complex into the h and declined thereafter. Intraportal injection of protamine/ cytosol from the endosomes (Zuidam and Barenholz, 1997). DOTAP/DNA led to about a 100-fold decrease in gene expression in the lung as compared with I.V. injection. 0092 Although cationic lipids have been used widely for Endothelial cells were the primary locus of lacZ transgene the delivery of genes, very few studies have used Systemic expression (Li and Huang, 1997). Protamine sulfate I.V. injection of cationic liposome-plasmid complexes. This enhanced plasmid delivery into Several different types of is because of the toxicity of the lipid component in animal cells in vitro, using the monovalent cationic liposomal models, not humans. Administration by I.V. injection of two formulations (DC-Chol and lipofectin). This effect was less types of cationic lipids of similar structure, DOTMA and pronounced with the multivalent cationic liposome formu DOTAP, shows that the transfection efficiency is determined lation, lipofectamine (Sorgi et al., 1997). mainly by the structure of the cationic lipid and the ratio of 0098 Spermine is found to enhance the transfection cationic lipid to DNA; the luciferase and GFP gene expres efficiency of DNA-cationic liposome complexes in cell Sion in different organs was transient, with a peak level culture and in animal Studies. This biogenic polyamine at between 4 and 24 hr, dropping to less than 1% of the peak high concentrations caused liposome fusion most likely level by day 4 (Song et al., 1997). promoted by the Simultaneous interaction of one molecule of Spermine (four positively charged amino groups) with the 0093. A number of different organs in vivo can be tar polar head groups of two or more molecules of lipids. At low geted after liposomal delivery of genes or oligonucleotides. concentrations (0.03-0.1 mM) it promoted anchorage of the Intravenous injection of cationic liposome-plasmid com liposome-DNA complex to the Surface of cells and enhanced plexes by tail vein in mice, targeted mainly the lung and to Significantly transfection efficiency (Boulikas, unpublished). a Smaller extent the liver, Spleen, heart, kidney and other organs (Zhu et al., 1993). Intraperitoneal injection of a 0099. The polycations polybrene, protamine, DEAE-dex plasmid-lipoSome complex expressing antisense K-ras RNA tran, and poly-L-lysine Significantly increased the efficiency in nude mice inoculated i.p. with ASPC-1 pancreatic cancer of adenovirus-mediated gene transfer in cell culture. This cells harboring K-ras point mutations and PCR analysis was thought to act by neutralizing the negative charges indicated that the injected DNA was delivered to various presented by membrane glycoproteins that reduce the effi organs except brain (Aoki et al., 1995). ciency of adenovirus-mediated gene transfer (Arcasoy et al., 1997). 0094) A number of factors for DOTAP:cholesterol/DNA 0100 Oligonucleotide Transfer complex preparation including the DNAlipoSome ratio, mild Sonication, heating, and extrusion were found to be 0101. In a further embodiment, the liposome encapsu crucial for improved Systemic delivery; maximal gene lates oligonucleotide DNA. Encapsulation of oligonucle expression was obtained when a homogeneous population of otides into liposomes increased their therapeutic index, DNA:liposome complexes between 200 to 450 nm in size prevented degradation in cultured cells, and in human Serum were used. Cryo-electron microscopy showed that the DNA and reduced toxicity to cells (Thierry and Dritschilo, 1992; was condensed on the interior of invaginated liposomes Capaccioli et al., 1993; Lewis et al., 1996). However, most between two lipid bilayers in these formulations, a factor Studies have been performed in cell culture, and very few in that was thought to be responsible for the high transfection animals in Vivo. There are still an important number of efficiency in vivo and for the broad tissue distribution improvements needed before these approaches can move (Templeton et al., 1997). into clinical Studies. 0102 Zelphati and Szoka (1997), have found that com 0.095 Steps to improve liposome-mediated gene delivery plexes of fluorescently labeled oligonucleotides with to Somatic cells include, persistence of the plasmid in blood DOTAP liposomes, entered the cell using an endocytic circulation, port of entry and transport acroSS the cell mem pathway mainly involving uncoated vesicles. Oligonucle brane, release from endoSomal compartments into the cyto otides were redistributed from punctate cytoplasmic regions plasm, nuclear import by docking through the pore com into the nucleus. This process was independent of acidifi plexes of the nuclear envelope, expression driven by the cation of the endoSomal vesicles. The nuclear uptake of appropriate promoter/enhancer control elements, and persis oligonucleotides depended on Several factors, Such as charge tence of the plasmid in the nucleus for long periods (Bou of the particle, where positively charged complexes were likas, 1998a). required for enhanced nuclear uptake. DOTAP increased US 2003/0072794 A1 Apr. 17, 2003 over 100 fold the antisense activity of a specific anti ment of blood circulation time of liposomes, by coating with luciferase oligonucleotide. Physicochemical Studies of oli PEG is described in U.S. Pat. No. 5,013,556. Typically, the gonucleotide-liposome complexes of different cationic lipid concentration of the PEG-modified phospholipids, or PEG compositions indicated that either phosphatidylethanola ceramide in the complex will be about 1-7%. In a particu mine or negative charges on other lipids in the cell mem larly preferred embodiment, the PEG-modified lipid is a brane are required for efficient fusion with cationic lipo PEG-DSPE. Some-oligonucleotide complexes to promote entry to the cell 0.108 Coating the surface of liposomes with inert mate (Jaaskelainen et al., 1994). rials designed to camouflage the liposome from the body's 0103). Similar results were reported by Lappalainen et al. host defense Systems was shown to increase remarkably the (1997). Digoxigenin-labeled oligodeoxynucleotides plasma longevity of liposomes. The biological paradigm for (ODNs) complexed with the polycationic DOSPA and the this “surface modified” Sub-branch was the erythrocyte, a monocationic DDAB (with DOPE as a helper lipid) were cell that is coated with a dense layer of carbohydrate groups, taken up by CaSki cells in culture by endocytosis. The and that manages to evade immune System detection and to nuclear membrane was found to pose a barrier against circulate for several months (before being removed by the nuclear import of ODNs that accumulated in the perinuclear same type of cell responsible for removing liposomes). area. Although DOSPA/DOPE liposomes could deliver ODNs into the cytosol, they were unable to mediate nuclear 0109 The first breakthrough came in 1987 when a gly import of ODNs. On the contrary, oligonucleotide-DDAB/ colipid (the brain tissue-derived ganglioside GM 1), was DOPE complexes with a net positive charge were released identified that, when incorporated within the lipid matrix, from Vesicles into the cytoplasm. It was determined that allowed liposomes to circulate for many hours in the blood DDAB/DOPE mediated nuclear import of the oligonucle stream (Allen and Chonn, 1987). A second glycolipid, otides. phosphatidylinositol, was also found to impart long plasma residence times to liposomes and, Since it was extracted 0104) DOPE-heme (ferric protoporphyrin IX) conju from Soybeans, not brain tissue, was believed to be a more gates, inserted in cationic lipid particles with DOTAP, pro pharmaceutically acceptable excipient (Gabizon et al., tected oligoribonucleotides from degradation in human 1989). Serum and increased oligoribonucleotide uptake into 2.2.15 human hepatoma cells. The enhancing effect of heme was 0110. A major advance in the surface-modified Sub evident only at a net negative charge in the particles (Takle branch was the development of polymer-coated liposomes et al., 1997). Uptake of liposomes labeled with 'In and (Allen et al. 1991). Polyethylene glycol (PEG) modification composed of DC-Chol and DOPE was primarily by liver, had been used for many years to prolong the half-lives of with Some accumulation in Spleen and skin and very little in biological proteins (such as enzymes and growth factors) the lung after I.V. tail injection. Preincubation of cationic and to reduce their immunogenicity (e.g. Beauchamp et al., liposomes with phosphorothioate oligonucleotide induced a 1983). It was reported in the early 1990s that PEG-coated dramatic, yet transient, accumulation of the lipid in lung that liposomes circulated for remarkably long times after intra gradually redistributed to liver. The mechanism of lung venous administration. Half-lives on the order of 24 h were uptake involved entrapment of large aggregates of oligo Seen in mice and rats, and over 30 hours in dogs. The term nucleotides within pulmonary capillaries at 15 min post "stealth' was applied to these liposomes because of their injection via embolism. Labeled oligonucleotide was local ability of evade interception by the immune system. The ized primarily to phagocytic vacuoles of Kupffer cells at 24 PEG hydrophilic polymers form dense “conformational h post-injection. Nuclear uptake of oligonucleotides in vivo clouds' to prevent other macromolecules from interaction was not observed (Litzinger et al., 1996). with the Surface, even at low concentrations of the protecting polymer (Gabizon and Papahadjopoulos, 1988; Papahad 0105 Polyethylene Glycol (PEG)-Coated Liposomes jopoulos et al., 1991; reviewed by Torchilin, 1998). The 0106. In a further embodiment, the liposome encapsu increased hydrophilicity of the liposomes after their coating lated DNA described herein, further comprise coating of the with the amphipathic PEG5000 leads to a reduction in final complex in step 2 (FIG. 1) with PEG. It is often nonspecific uptake by the reticuloendothelial System. desirable to conjugate a lipid to a polymer that confers 0111 Whereas the half-life of antimyosin immunolipo extended half-life, such as polyethylene glycol (PEG). somes was 40 min, by coating with PEG, they increased Derivatized lipids that are employed, include PEG-modified their half-life to 1000 min after intravenous injection to DSPE or PEG-ceramide. Addition of PEG components prevents complex aggregation, increases circulation lifetime rabbits (Torchilin et al., 1992). of particles (liposomes, proteins, other complexes, drugs) 0112 Micelles, Surfactants and Small Unilamellar and increases the delivery of lipid-nucleic acid complexes to Vesicles the target tissues. See, Maxfield et al., Polymer 16:505-509 0113. In a further embodiment, the liposome encapsu (1975); Bailey, F. E. et al., in: Nonionic Surfactants, Schick, lated DNA described herein, further comprise an initial step M.J., ed., pp. 794-821 (1967); Abuchowski, A. et al., J. Biol. of micelle formation between cationic lipids and condensed Chem. 252:3582-3586 (1977); Abuchowski, A. et al., Can plasmid or oligonucleotide DNA in ethanol Solutions. cer Biochem. Biophys. 7:175-186 (1984); Katre, N. V. et al., Micelles are small amphiphilic colloidal particles formed by Proc. Natl. Acad. Sci. USA 84:1487-1491 (1987); Goodson, certain kinds of lipid molecules, detergents or Surfactants R. et al. Bio Technology 8:343-346 (1990). under defined conditions of concentration, Solvent and tem 0107 Conjugation to PEG is reported to have reduced perature. They are composed of a Single lipid layer. Micelles immunogenicity and toxicity. See, Abuchowski et al., J. can have their hydrophilic head groups assembled exposing Biol. Chem. 252:3578-3581 (1977). The extent of enhance their hydrophobic tails to the solvent (for example in US 2003/0072794 A1 Apr. 17, 2003

30-60% aqueous ethanol solution) or can reverse their nuclear localization of the cationic lipids (along with plas Structures exposing their polar heads toward the Solvent Such mid DNA) to the nucleus. Indeed, a number of studies as by lowering the concentration of the ethanol to below Suggest that plasmid DNA is imported to nuclei; its trans 10% (reverse micelles). Micelle systems are in thermody location docks cationic lipid molecules electroStatically namic equilibrium with the Solvent molecules and environ attached to the DNA. These cationic lipid molecules exert ment. This results in constant phase changes, especially their toxicity by interfering with the nucleoSome and domain upon contact with biological materials, Such as upon intro Structure of the chromatin causing local destabilization. This duction to cell culture, injection to animals, dilution, contact disturbance or aberrant chromatin reorganization could be with proteins or other macromolecules. These changes result exerted at the level of the nuclear matrix where plasmid in rapid micelle disassembly or flocculation. This is in DNA is attached for transcription, autonomous replication, contrast to the much higher Stability of lipoSome bilayers. or integration via recombination. 0114. Single-chain surfactants are able to form micelles 0117 Surfactants have found wide application in formu (see Table 1, below). These include the anionic (sodium lations such as emulsions (including microemulsions) and dodecyl Sulfate, cholate or oleate) or cationic (cetyl-trim liposomes. The most common way of classifying and rank ethylammonium bromide, CTAB) Surfactants. CTAB, ing the properties of the many different types of Surfactants, CTAC, and DOIC micelles yielded larger solubility gaps both natural and synthetic, is by the use of the hydrophile/ (lower concentration of colloidally suspended DNA) than lipophile balance (HLB). The use of Surfactants in drug corresponding SUV particles containing neutral lipid and products, formulations and in emulsions has been reviewed CTAB (1:1) (Lasic, 1997). (Rieger, in: Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, 1988, p. 285). TABLE 1. 0118 Nonionic Surfactants find wide application in phar Molecules able to form micelles maceutical and cosmetic products and are uSable over a wide range of pH values. In general, their HLB values range from Molecule Reference 2 to about 18, depending on their structure. Nonionic CTAB, CTAC, DOIC Lasic, 1997 Surfactants include, nonionic esterS Such as ethylene glycol Detergent/phospholipid micelles Lusa et al., "1998 esters, propylene glycol esters, glyceryl esters, polyglyceryl Dodecyl betaine (amphoteric surfactant) de la Maza et al., 1998 esters, Sorbitan esters, Sucrose esters, and ethoxylated esters. Dodecylphosphocholine cholate Lasic, 1997 Glycine-conjugated bile salt (anionic Leonard and Cohen, 1998 Nonionic alkanolamides and ethers, Such as fatty alcohol steroid detergent-like molecule) ethoxylates, propoxylated alcohols, and ethoxylated/pro Lipid-dodecyl maltoside micelles Lambert et al., 1998 poxylated, block polymers are also included in this class. mixed micelles (Triton X-100 & Lopez et al., 1998 The polyoxyethylene Surfactants are the most popular mem phosphatidylcholine) bers of the nonionic Surfactant class. Octylglucoside (non-ionic straight Leonard and Cohen, 1998 chain detergent) Oleate Lasic, 1997 0119) Anionic surfactants include carboxylates such as PEG-dialkylphosphatidic Tirosh et al., 1998 Soaps, acyl lactylates, acyl amides of amino acids, esters of acid (dihexadecylphosphatidyl Sulfuric acid Such as alkyl Sulfates and ethoxylated alkyl (DHP)-PEG2000) Sulfates, Sulfonates Such as alkyl benzene Sulfonates, acyl Phosphatidylcholine (neutral Zwitterionic) Schroeder et al., 1990 Polyethyleneglycol (MW 5000)-distearoyl Weissig et al., 1998 isethionates, acyl taurates and SulfoSuccinates, and phos phosphatidyl ethanolamine (PEG-DSPE) phates. The most important members of the anionic Surfac sodium dodecyl sulfate (anionic straight Leonard and Cohen, 1998 tant class are the alkyl Sulfates and the Soaps. chain detergent) Sodium taurofusidate (conjugated fungal Leonard and Cohen, 1998 0120 Cationic surfactants include quaternary ammonium bile salt analog) Salts and ethoxylated amines. The quaternary ammonium Taurine-conjugated bile salts (anionic Leonard and Cohen, 1998 salts are the most used members of this class. If the steroid detergent-like molecule) Triton X-100 surfactant Surfactant molecule has the ability to carry either a positive Lasic, 1997 or negative charge, the Surfactant is classified as amphoteric. Amphoteric Surfactants include acrylic acid derivatives, 0115 There is a critical detergent/phospholipid ratio at Substituted alkylamides, N-alkylbetaines and phosphatides. which lamellar-to-micellar transition occurs. For example, 0121 Classical micelles may not be effective as gene the vesicle-micelle transition was observed for dodecyl transfer vehicles, but important intermediates in the forma maltoside with large unilamellar liposomes. A Striking fea tion of lipoSome complexes encapsulating drugs or nucleic ture of the Solubilization proceSS by dodecyl maltoside was acids. The stability of single chain surfactants-DNA-colloi the discovery of a new phase, consisting of a very Viscous dal Systems is lower than SUV particles containing neutral 'gel-like' Structure composed of long filamentous thread lipid and CTAB (1:1). However, second generation micelles like micelles, over 1 to 2 microns in length. are able to target tumors in Vivo. Weissig and co-workers 0116. A long circulating complex needs to be slightly (1998) used the soybean trypsin inhibitor (STI) as a model anionic. Therefore the liposomes used for the conversion of protein to target tumors. STI was modified with a hydro the micelles into liposomes contain bipolar lipids (PC, PE) phobic residue of N-glutaryl-phosphatidyl-ethanolamine and 1-30% negatively charged lipids (DPPG). The cationic (NGPE) and incorporated into both polyethyleneglycol lipids which are toxic, are hidden in the inner lipoSome (MW 5000)-distearoyl phosphatidyl ethanolamine (PEG membrane bilayer. Those reaching the solid tumor will exert DSPE) micelles (<20 nm) and PEG-DSPE-modified long their toxic effects causing apoptosis. Apoptosis will be circulating liposomes (ca. 100 um). AS determined from the caused by the delivery of the toxic drug or anti-neoplastic protein label by using illn attached to Soybean trypsin gene or oligonucleotide to the cancer cell but also by the inhibitor via protein-attached diethylene triamine pentaace US 2003/0072794 A1 Apr. 17, 2003

tic acid, DTPA, PEG-lipid micelles accumulated better than HA-chimeras were designed in which the cytoplasmic tail the same protein anchored in long-circulating PEG-lipo and/or transmembrane domain of HA was replaced with the Somes in Subcutaneously established Lewis lung carcinoma corresponding domains of the fusogenic glycoprotein F of in mice after tail vein injection. Sendai virus. Constructs of HA were made in which the 0.122 Loading a liposomal dispersion with an cytoplasmic tail was replaced by peptides of human neu rofibromin type 1 (NF1) (residues 1441 to 1518) or c-Raf-1, amphiphilic drug may cause a phase transformation into a (residues 51 to 131) and were expressed in CV-1 cells by micellar Solution. The transition from high ratios of phos using the vaccinia virus-T7 polymerase transient-expression pholipid to drug (from 2:1 to 1:1 downwards) were accom system. Membrane fusion between CV-1 cells and bound panied by the conversion of liposomal dispersions of milky human erythrocytes (RBCS) mediated by parental or chi white appearance (particle size 200 m) to nearly transparent meric HA proteins showed that, after the pH was lowered, a micelles (particle size below 25 nm). See, Schutze and flow of the aqueous fluorophore calcein from preloaded Muller-Goymann (1998). RBCs into the cytoplasm of the protein-expressing CV-1 0123 Fusogenic Peptides cells took place. This indicated that membrane fusion 0.124. In a further embodiment, the liposome encapsu involves both leaflets of the lipid bilayers and leads to lated DNA described herein further comprises an effective formation of an aqueous fusion pore (Schroth-Diaz et al., amount of a fusogenic peptide. Fusogenic peptides belong to 1998). a class of helical amphipathic peptides characterized by a 0128. A remarkable discovery was that the TAT protein of hydrophobicity gradient along the long helical axis. This HIV is able to cross cell membranes (Green and Loewen hydrophobicity gradient causes the tilted insertion of the stein, 1998) and that a 36-amino acid domain of TAT, when peptides in membranes, thus destabilizing the lipid core and, chemically croSS-linked to heterologous proteins, conferred thereby, enhancing membrane fusion (Decout et al., 1999). the ability to transduce into cells. The 11-amino acid fuso 0125 Hemagglutinin (HA) is a homotrimeric surface genic peptide of TAT (YGRKKRRQRRR (SEQ ID NO:10)) glycoprotein of the influenza virus. In infection, it induces is a nucleolar localization signal (see Boulikas, 1998b). membrane fusion between Viral and endoSomal membranes at low pH. Each monomer consists of the receptor-binding 0129. Another protein of HIV, the glycoprotein gp41, HA1 domain and the membrane-interacting HA2 domain. contains fuSogenic peptides. Linear peptides derived from The NH2-terminal region of the HA2 domain (amino acids the membrane proximal region of the gp41 ectodomain have 1 to 127), the so-called “fusion peptide,” inserts into the potential applications as anti-HIV agents and inhibit infec target membrane and playS a crucial role in triggering fusion tivity by adopting a helical conformation (Judice et al., between the viral and endosomal membranes. Based on the 1997). The 23 amino acid residue, N-terminal peptide of Substitution of eight amino acids in region 5-14 with cyS HIV-1 gp41 has the capacity to destabilize negatively teines and spin-labeling electron paramagnetic resonance, it charged large unilamellar vesicles. In the absence of cations, was concluded that the peptide forms an alpha-helix tilted the main Structure was a pore-forming alpha-helix, whereas approximately 25 degrees from the horizontal plane of the in the presence of Ca" the conformation switched to a membrane with a maximum depth of 15 A from the phos fuSogenic, predominantly extended beta-type Structure. The phate group (Macosko et al., 1997). Use of fusogenic fusion activity of HIV(ala) (bearing the R22->A substitu peptides from influenza virus hemagglutinin HA-2 enhanced tion) was reduced by 70%, whereas fusogenicity was com greatly the efficiency of transferrin-polylysine-DNA com pletely abolished when a second substitution (V2->E) was plex uptake by cells. The peptide was linked to polylysine included, arguing that it is not an alpha-helical but an and the complex was delivered by the transferrin receptor extended structure adopted by the HIV-1 fusion peptide that mediated endocytosis (reviewed by Boulikas, 1998a). This actively destabilizes cholesterol-containing, electrically peptide has the sequence: GLFEAIAGFI ENGWEGMIDG neutral membranes (Pereira et al., 1997). GGYC (SEQ ID NO:9) and is able to induce the release of 0130. The prion protein (PrP) is a glycoprotein of the fluorescent dye calcein from liposomes prepared with unknown function normally found at the Surface of neurons egg yolk phosphatidylcholine, which was higher at acidic and of glial cells. It is involved in diseases Such as bovine pH. This peptide was also able to increase up to 10-fold the Spongiform encephalopathy, and Creutzfeldt-Jakob disease anti-HIV potency of antisense oligonucleotides, at a con in humans, where PrP is converted into an altered form centration of 0.1-1 mM, using CEM-SS lymphocytes in (termed PrPSc). According to computer modeling calcula culture. This peptide changes conformation at the slightly tions, the 120 to 133 and 118 to 135 domains of PrP are tilted more acidic environment of the endoSome, destabilizing and lipid-associating peptides inserting in a oblique way into a breaking the endoSomal membrane (reviewed by Boulikas, lipid bilayer and able to interact with liposomes to induce 1998a). leakage of encapsulated calcein (Pillot et al., 1997b). 0.126 The presence of negatively charged lipids in the 0131 The C-terminal fragments of the Alzheimer amy membrane is important for the manifestation of the fuSO loid peptide (amino acids 29-40 and 29-42) have properties genic properties of Some peptides, but not of others. related to those of the fusion peptides of Viral proteins Whereas the fusogenic action of a peptide, representing a inducing fusion of lipoSomes in Vitro. These properties could putative fusion domain of fertilin, a Sperm Surface protein mediate a direct interaction of the amyloid peptide with cell involved in Sperm-egg fusion, was dependent upon the membranes and account for part of the cytotoxicity of the presence of negatively charged lipids, that of the HIV2 amyloid peptide. In View of the epidemiologic and bio peptide was not (Martin and Ruysschaert, 1997). chemical linkages between the pathology of Alzheimer's 0127. For example, to analyze the two domains on the disease and apolipoprotein E (apo) polymorphism, exami fuSogenic peptides of influenza virus hemagglutinin HA, nation of the potential interaction between the three common US 2003/0072794 A1 Apr. 17, 2003 13 apoE isoforms and the C-terminal fragments of the amyloid tion in lipid bilayers in membranes of cells or lipoSomes is peptide showed that only apoE2 and apoE3, not apoE4, are responsible for the membrane fusion properties of peptides. potent inhibitors of the amyloid peptide fusogenic and aggregational properties. The protective effect of apoE Zn", enhances the fusogenic activity of peptides because it against the formation of amyloid aggregates was thought to Stabilizes the alpha-helical Structure. For example, the be mediated by the formation of stable apoE/amyloid pep HEXXH (SEQ ID NO:11) domain of the salivary antimi tide complexes (Pillot et al., 1997a; Lins et al., 1999). crobial peptide, located in the C-terminal functional domain 0132) The fusogenic properties of an amphipathic net of histatin-5, a recognized zinc-binding motif is in a heli negative peptide (WAE 11), consisting of 11 amino acid coidal conformation (Martin et al., 1999; Melino et al., residues were Strongly promoted when the peptide was 1999; Curtain et al., 1999). anchored to a liposomal membrane. The fusion activity of the peptide appeared to be independent of pH and membrane 0134) Fusion peptides have been formulated with DNA merging, and the target membranes required a positive plasmids to create peptide-based gene delivery Systems. A charge that was provided by incorporating lysine-coupled combination of the YKAKnWK (SEQ ID NO:12) peptide, phosphatidylethanolamine (PE-K). Whereas the coupled used to condense plasmids into 40 to 200 nm nanoparticles, peptide could cause vesicle aggregation via nonspecific with the GLFEALLELLESLWELLLEA (SEQ ID NO: 13) electrostatic interaction with PE-K, the free peptide failed to amphipathic peptide, that is a pH-sensitive lytic agent induce aggregation of PE-K vesicles (Pecheur et al., 1997). designed to facilitate release of the plasmid from endoSomes 0133) A number of studies suggest that stabilization of an enhanced expression Systems containing the beta-galactosi alpha-helical Secondary Structure of the peptide after inser dase reporter gene (Duguid et al., 1998). See Table 2, below.

TABLE 2 Fusogenic peptides Fusogenic peptide Source Protein Properties Reference GLFEAIAGFENGWEG 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 70 residue peptide (SV Fusion peptide Induced lipid mixing of egg Ghosh and Shai, 117) and N-terminal phosphatidylcholine 1999 heptad repeat of phosphatidylglycerol 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 ipid bilayer and induced Virus (DHBV) eakage of internal aqueous contents from both neutral and negatively charged iposomes MSPSSLLGLLAGLOVV S protein of Was inserted into the Rodriguez-Crespo et (SEQ ID NO:14) woodchuck hydrophobic core of the al., 1999 hepatitis B virus ipid bilayer and induced (WHV) eakage of internal aqueous contents from both neutral and negatively charged iposomes N-terminus of Nef Nef protein of Membrane-perturbing and Macreadie et al., human fusogenic activities in 1997 inimuno artificial membranes; causes deficiency cell killing in E. coli and type 1 (HIV-1) yeast Amino-terminal sequence F1 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 US 2003/0072794 A1 Apr. 17, 2003 14

TABLE 2-continued Fusogenic peptides Fusogenic peptide Source Protein Properties Reference iposomes 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 ilted fragment of viral usion proteins Non-aggregated amyloid Alzheimer's beta Induces apoptotic neuronal Pillot et al., 1999 beta-peptide (1-40) amyloid peptide cell death LCAT 56-68 helical Lecithin Forms stable beta-sheets in Peelman et al., 1999; segment cholesterol ipids Decout et al., 1999 acyltransferase (LCAT) Peptide sequence B18 Membrane Triggers fusion between Ulrich et al., 1999 associated sea ipid vesicles; a histidine urchin sperm nch motif for binding zinc protein binding is required for the fusogenic unction 53-70 (C-terminal helix) Apollipoprotein Induces fusion of Lambert et al., 1998 (apo) AII unilamellar lipid vesicles and displaces apo AI from HDL and r-HDL Residues 90-111 PH-30 alpha (a Membrane-fusogenic Nudome 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 dimyristoylphosphatidyl glycerol 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 Histatin-5 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) usion 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) he membrane (favored at 37 C.) oriented almost parallel to the lipid acyl chains; promotes fusion of arge 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 he imidazole groups at pH residues below 6.0.; disrupt endosomal membranes GLFEALLELLESLWELL Synthetic Amphipathic peptide; a pH Duguid et al., 1998 LEA (SEQ ID NO:4) sensitive lytic agent to acilitate release of the plasmid from endosomes (LKKL) (SEQ ID NO: 15) Synthetic Amphiphilic fusogenic Gupta and Kothekar, peptide, able to interact with 1997 Our molecules of DMPC Ac-(Leu-Ala-Arg-Leu)- Synthetic; basic Caused a leakage of Suenaga et al., 1989; NHCH (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) US 2003/0072794 A1 Apr. 17, 2003 15

TABLE 2-continued Fusogenic peptides Fusogenic peptide Source Protein Properties Reference Amphiphilic anionic Synthetic Can mimic the fusogenic Murata et al., 1991 peptides E5 and E5L activity of influenza hemagglutinin (HA) 30-amino acid peptide with Synthetic; Becomes an amphipathic Parente et al., 1988 the major repeat unit Glu- designed alpha-helix as the pH is Ala-Leu-Ala (GALA), to mimic lowered to 5.0; fusion of (SEQ ID NO:17) the behavior of phosphatidylcholine small the fusogenic unilamellar vesicles induced sequences of viral by GALA requires a peptide fusion proteins 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 alpha represents 2 helix; caused fusion of aminoisobutyric acid EYPC liposomes and dipalmitoylphosphatidylcholine liposomes more strongly with decreasing pH

0135 Fusogenic Lipids In contrast to protein transcription factors that are exclu 0.136 DOPE is a fusogenic lipid; elastase cleavage of Sively nuclear possessing Strong karyophilic peptides com N-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE (SEQ ID posed of at least four arginines, (R), and lysines, (K), within NO:19) converted this derivative to DOPE (overall positive an hexapeptide flanked by proline and glycine helix-break charge) to deliver an encapsulated fluorescent probe, cal ers, protein kinases often contain one histidine and three cein, into the cell cytoplasm (Pak et al., 1999). An oligode K+R residues (Boulikas, 1996). This was proposed to Oxynucleic Sequence of 30 bases complementary to a region Specify a weak NLS Structure resulting in the nuclear import of beta-endorphin mRNA elicited a concentration-dependent of a fraction of the total cytoplasmic kinase molecules, as inhibition of beta-endorphin production in cell culture after well as in their weak retention in the different ionic Strength it was encapsulated within small unilamellar vesicles (50 nuclear environment. Putative NLS peptides in protein nm) containing dipalmitoyl-DL-alpha-phosphatidyl-L- kinases may also contain hydrophobic or bulky aromatic Serine endowed with fusogenic properties (Fresta et al., amino acids proposed to further diminish their capacity to 1998). act as Strong NLS. 0140 Most mammalian proteins that participate in DNA 0137) Nuclear Localization Signals (NLS) repair pathways seem to possess Strong karyophilic clusters 0.138. In a further embodiment, the liposome encapsu containing at least four R+K Over a stretch of six amino lated plasmid or oligonucleotide DNA described herein acids (Table 7). further comprise an effective amount of nuclear localization 0141 Rules to predict nuclear localization of an unknown Signal (NLS) peptides. Trafficking of nuclear proteins from protein Several Simple rules have been proposed for the the Site of their Synthesis in the cytoplasm to the Sites of prediction of the nuclear localization of a protein of an function in the nucleus through pore complexes is mediated unknown function from its amino acid Sequence: by NLSs on proteins to be imported into nuclei (Tables 3-10, below). Protein translocation from the cytoplasm to the 0142 (i) An NLS is defined as four arginines (R) plus nucleoplasm involves: (i) the formation of a complex of lysines (K) within an hexapeptide; the presence of one or karyopherin C. with NLS-protein; (ii) Subsequent binding of more histidines (H) in the tetrad of the karyophilic hexapep karyopherin B; (iii) binding of the complex to FXFG peptide tide, often found in protein kinases that have a cytoplasmic repeats on nucleoporins; (iv) docking of Ran-GDP to nucle and a nuclear function, may specify a weak NLS whose oporin and to karyopherin heterodimer by p10, (v) a number function might be regulated by phosphorylation or may of association-dissociation reactions on nucleoporins that Specify proteins that function in both the cytoplasm and the dock the import Substrate toward the nucleoplasmic side nucleus (Boulikas, 1996); with a concomitant GDP-GTP exchange reaction transform 0143 (ii) The K/R clusters are flanked by the a-helix ing Ran-GDP into Ran-GTP and catalyzed by karyopherin breakers G and P thus placing the NLS at a helix-turn-helix C.; and (vi) dissociation from karyopherin B and release of or end of a a-helix. Negatively-charged amino acids (D, E) the karyopherin C/NLS-protein by Ran-GTP to the nucleo are often found at the flank of the NLS and on Some plasm. occasions may interrupt the positively-charged NLS cluster; 0139 Karyophilic and acidic clusters were found in most 0144 (iii) Bulky amino acids (W, F, Y) are not present non-membrane Serine/threonine protein kinases whose pri within the NLS hexapeptide; mary structure has been examined (Table 6). These karyo 0145 (iv) NLS signals may not be flanked by long philic clusters might mediate the anchoring of the kinase Stretches of hydrophobic amino acids (e.g. five); a mixture molecules to transporter proteins for their regulated nuclear of charged and hydrophobic amino acids Serves as a mito import and might constitute the nuclear localization signals. chondrial targeting Signal; US 2003/0072794 A1 Apr. 17, 2003

0146 (v) The higher the number of NLSs, the more important implications for human disease. Both nuclear readily a molecule is imported to the nucleus (Dworetzky et import and export processes can be manipulated by conju al., 1988). Even Small proteins, for example histones (10-22 gation of proteins with NLS or NES peptides. During gene kDa), need to be actively imported to increase their import therapy, the foreign DNA needs to enter nuclei for its rates compared with the slow rate of diffusion of small transcription. A pathway is proposed involving the complex molecules through pores, ation of plasmids and oligonucleotides with nascent nuclear proteins possessing NLSS as a prerequisite for their nuclear 0147 (vi) Signal peptides are stronger determinants than import. Covalent linkage of NLS peptides to oligonucle NLSS for protein trafficking. Signal peptides direct proteins otides and plasmids or formation of complexes of plasmids to the lumen of the endoplasmic reticulum for their Secretion with proteins possessing multiple NLS peptides was pro or insertion into cellular membranes (presence of transmem posed (Boulikas, 1998b) to increase their import rates and brane domains) (Boulikas, 1994); the efficiency of gene expression. Cancer cells were pre 0148 (vii) Signals for the mitochondrial import of pro dicted to import more efficiently foreign DNA into nuclei, teins (a mixture of hydrophobic and karyophilic amino compared with terminally differentiated cells because of acids) may antagonize nuclear import Signals and proteins their increased rates of proliferation and protein import. possessing both type of Signals may be translocated to both 0154 Antineoplastic Drugs mitochondria and nuclei; O155 In a further embodiment, the liposome encapsu 0149 (viii) Strong association of a protein with large lated plasmid or oligonucleotide DNA described herein, cytoplasmic structures (membrane proteins, intermediate further comprises its use for reducing tumor size or restrict filaments) make Such proteins unavailable for import even ing its growth with combination with encapsulated or free though they posses NLS-like peptides (Boulikas, 1994); antineoplastic agents. Antineoplastic agents preferably are: (i) alkylating agents having the bis-(2-chloroethyl)-amine 0150 (ix). Transcription factors and other nuclear pro group Such as chlormethine, chlorambucile, melphalan, ura teins posses a great different number of putative NLS mustine, mannomustine, extramustinephosphat, mechlore stretches. Of the sixteen possible forms of putative NLS thaminoxide, cyclophosphamide, ifosfamide, or trifosfa structures the most abundant types are the 00x00, 000x0, mide; (ii) alkylating agents having a Substituted aziridine 0000, and 00x0x0, where 0 is R or K, together accounting group, for example tretamine, thiotepa, triaziquone, or mito for about 70% of all karyophilic clusters on transcription mycine; (iii) alkylating agents of the methaneSulfonic ester factors (Boulikas, 1994); type Such as buSulfane; (iv) alkylating N-alkyl-N-ni 0151 (x) A small number of nuclear proteins seem to be troSourea derivatives, for example carmustine, lomustine, void of a typical karyophilic NLS. Either non karyophilic Semustine, or Streptozotocine, (v) alkylating agents of the peptides function for their nuclear import, as Such molecules mitobronitole, dacarbazine, or procarbazine type; (vi) com possess bipartite NLSs, or these NLS-less proteins depend plexing agents Such as cis-platin; (vii) antimetabolites of the absolutely for import on their Strong complexation in the folic acid type, for example methotrexate, (viii) purine cytoplasm with a nuclear protein partner able to be imported derivatives Such as mercaptopurine, thioguanine, azathio (Boulikas, 1994). This mechanism may ensure a certain prine, tiamiprine, Vidarabine, or puromycine and purine Stoichiometric ratio of the two molecules in the nucleus, and nucleoside phosphorylase inhibitors; (ix) pyrimidine deriva might be of physiological significance; and tives, for example fluorouracil, floXuridine, tegafur, cytara bine, idoxuridine, flucytosine; (x) antibiotics Such as dacti 0152 (xi) A number of proteins may be imported via nomycin, daunorubicin, doxorubicin, mithramycin, other mechanisms not dependent on classical NLS. bleomycin or etoposide; (xi) Vinca alkaloids; (xii) inhibitors 0153. A number of processes have been found to be of proteins overexpressed in cancer cells Such as telomerase regulated by nuclear import including nuclear translocation inhibitors, glutathione inhibitors, proteasome inhibitors, of the transcription factors NF-KB, rNFIL-6, ISGF3, SRF, (xiii) modulators or inhibitors of Signal transduction path c-Fos, GR as well as human cyclins A and BI, casein kinase ways Such as phosphatase inhibitors, protein kinase C II, cAMP-dependent protein kinase II, protein kinase C, inhibitors, casein kinase inhibitors, insulin-like growth fac ERKI and ERK2. Failure of cells to import specific proteins tor-1 receptor inhibitor, ras inhibitors, ras-GAP inhibitor, into nuclei can lead to carcinogenesis. For example, BRCA1 protein tyrosine phosphatase inhibitors, (xiv) tumor angio is mainly localized in the cytoplasm in breast and ovarian genesis inhibitorS Such as angiostatin, oncostatin, endostatin, cancer cells, whereas in normal cells the protein is nuclear. thalidomide, (XV) modulators of the immune response and mRNA is exported through the same route as a complex with cytokines Such as interferons, interleukins, TNF-alpha; (XVi) nuclear proteins possessing nuclear export Signals (NES). modulators of the extracellular matrix Such as matrix met The majority of proteins with NES are RNA-binding pro alloproteinase inhibitors, Stromelysin inhibitors, plasmino teins that bind to and escort RNAS to the cytoplasm. gen activator inhibitor; (XVii) hormone modulators for hor However, other proteins with NES function in the export of mone-dependent cancers (breast cancer, prostate cancer) proteins; CRM1, that binds to the NES sequence on other Such as antiandrogen, estrogens, (XViii) apoptosis regulators; proteins and interacts with the nuclear pore complex, is an (xix) bFGF inhibitor; (xx) multiple drug resistance gene essential mediator of the NES-dependent nuclear export of inhibitor; (XXi) monoclonal antibodies or antibody frag proteins in eukaryotic cells. Nuclear localization and export ments against antigenes overexpressed in cancer cells (anti signals (NLS and NES) are found on a number of important Her2/neu for breast cancer); (XXii) anticancer genes whose molecules, including p53, V-Rel, the transcription factor expression will cause apoptosis, arrest the cell cycle, induce NF-ATc, the c-Ab1 nonreceptor tyrosine kinase, and the an immune response against cancer cells, inhibit tumor fragile X Syndrome mental retardation gene product. The angiogenesis i.e. formation of blood vessels, tumor Suppres deregulation of their normal import/export trafficking has sor genes (p53, RB, BRCA1, E1A, bc1-2, MDR-1, p21, p16, US 2003/0072794 A1 Apr. 17, 2003

bax, bcl-XS, E2F, IGF-I VEGF, angiostatin, oncostatin, tard, uredepa, Vapreotide, verteporfin, vinblastine Sulfate, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-y, and Vincristine Sulfate, Vindesline, Vindesine Sulfate, Vinepidine TNF-a); and (XXiii) antisense oligonucleotides (antisense Sulfate, Vinglycinate Sulfate, Vinleurosine Sulfate, Vinorel c-fos, c-myc, K-ras). Optionally these drugs are adminis bine tartrate, Vinrosidine Sulfate, Vinzolidine Sulfate, Voro tered in combination with chlormethamine, prednisolone, Zole, Zeniplatin, Zinostatin, Zorubicin hydrochloride. prednisone, or procarbazine or combined with radiation therapy. Future new anticancer drugs added to the arsenal O157. Other anti-cancer drugs include: 20-epi-1,25 dihy are expected to be ribozymes, triplex-forming oligonucle droxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, otides, gene inactivating oligonucleotides, a number of new acylfulvene, adecypenol, adoZelesin, aldesleukin, ALL-TK genes directed against genes that control the cell prolifera antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, tion or Signaling pathways, and compounds that block signal anastrozole, andrographolide, angiogenesis inhibitors, transduction. antagonist D, antagonist G, antarelix, anti-dorsalizing mor 0156 Anti-cancer drugs include: acivicin, aclarubicin, phogenetic protein-1, antiandrogen, antiestrogen, antine acodazole hydrochloride, acronine, adoZelesin, adriamycin, oplaston, antisense oligonucleotides, aphidicolin glycinate, aldesleukin, altretamine, ambomycin, ametantrone acetate, apoptosis gene modulators, apoptosis regulators, apurinic aminoglutethimide, amsacrine, anastrozole, anthramycin, acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, asparaginase, asperlin, azacitidine, azetepa, azotomycin, atameStane, atrimustine, axinastatin 1, axinastatin 2, axin batimastat, benzodepa, bicalutamide, bisantrene hydrochlo astatin 3, aZasetron, azatoxin, azatyrosine, baccatin III ride, bisnafide dimeSylate, bizelesin, bleomycin Sulfate, bre derivatives, balanol, batimastat, BCR/ABL antagonists, ben quinar Sodium, bropirimine, buSulfan, cactinomycin, calus Zochlorins, benzoylstaurosporine, beta lactam derivatives, terone, caracemide, carbetimer, carboplatin, carmustine, beta-alethine, betaclamycin B, betulinic acid, bFGF inhibi carubicin hydrochloride, carZelesin, cedefingol, chloram tor, bicalutamide, bisantrene, bisaZiridinylspermine, bisna bucil, cirolemycin, cisplatin, cladribine, crisinatol meSylate, fide, bistratene A, bizelesin, breflate, bropirimine, budoti cyclophosphamide, cytarabine, dacarbazine, dactinomycin, tane, buthionine Sulfoximine, calcipotriol, callphostin C, daunorubicin hydrochloride, decitabine, deXormaplatin, camptothecin derivatives, canarypox IL-2, capecitabine, car deZaguanine, deZaguanine meSylate, diaziquone, docetaxel, boxamide-amino-triazole, carboxyamidotriazole, CaRest doxorubicin, doxorubicin hydrochloride, droloxifene, M3, CARN 700, cartilage derived inhibitor, carzelesin, droloxifene citrate, dromoStanolone propionate, duaZomy casein kinase inhibitors (ICOS), castanospermine, cecropin cin, edatrexate, eflomithine hydrochloride, elsamitrucin, B, cetrorelix, chlorlns, chloroquinoxaline Sulfonamide, cica enloplatin, enpromate, epipropidine, epirubicin hydrochlo prost, cis-porphyrin, cladribine, clomifene analogues, clot ride, erbulozole, eSorubicin hydrochloride, estramustine, rimazole, collismycin A, collismycin B, combretastatin A4, estramustine phosphate Sodium, etanidazole, etoposide, eto combretastatin analogue, conagenin, crambescidin 816, cri poside phosphate, etoprine, fadrozole hydrochloride, faZara Snatol, cryptophycin 8, cryptophycin A derivatives, curacin bine, fenretinide, floxuridine, fludarabine phosphate, fluo A, cyclopentanthraquinones, cycloplatam, cypemycin, cyt rouracil, flurocitabine, foSquidone, fostriecin Sodium, arabine ocfosfate, cytolytic factor, cytostatin, dacliximab, gemcitabine, gemcitabine hydrochloride, hydroxyurea, ida decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, rubicin hydrochloride, ifosfamide, ilmofosine, interferon deXraZOxane, deXVerapamil, diaziquone, didemnin B, didox, alfa-2a, interferon C-2b, interferon C-n1, interferon C.-n3, diethylnorspermine, dihydro-5-aZacytidine, dihydrotaXol, interferon B-ia, interferon Y-ib, iproplatin, irinotecan hydro 9-dioxamycin, diphenyl Spiromustine, docosanol, dolas chloride, lanreotide acetate, letrozole, leuprolide acetate, etron, doxifluridine, droloxifene, dronabinol, duocarmycin liarozole hydrochloride, lometrexol Sodium, lomustine, SA, ebSelen, ecomustine, edelfosine, edrecolomab, eflorni loSOXantrone hydrochloride, masoprocol, maytansine, thine, elemene, emitefur, epirubicin, epristeride, estramus mechlorethamine hydrochloride, megestrol acetate, tine analogue, estrogen agonists, estrogen antagonists, melengestrol acetate, melphalan, menogaril, mercaptopu etanidazole, etoposide phosphate, exemestane, fadrozole, rine, methotrexate, methotrexate Sodium, metoprine, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, meturedepa, mitindomide, mitocarcin, mitocromin, mitogil fleZelastine, fluiasterone, fludarabine, fluorodaunorunicin lin, mitomalcin, mitomycin, mitosper, mitotane, mitox hydrochloride, forfenimex, formeStane, fostriecin, fotemus antrone hydrochloride, mycophenolic acid, nocodazole, tine, gadolinium gallium nitrate texaphyrin, galocitabine, nogalamycin, Ormaplatin, OXiSuran, paclitaxel, pegaspar ganirelix, gelatinase inhibitors, gemcitabine, glutathione gase, peliomycin, pentamustine, peplomycin Sulfate, perfoS inhibitors, hepSulfam, heregulin, hexamethylene bisaceta famide, pipobroman, pipOSulfan, piroXantrone hydrochlo mide, hypericin, ibandronic acid, idarubicin, idoxifene, idra ride, plicamycin, plomeStane, porfimer Sodium, mantone, ilmofoSine, illomastat, imidazoacridones, imiqui porfiromycin, prednimustine, prednisone, procarbazine mod, immunostimulant peptides, insulin-like growth hydrochloride, puromycin, puromycin hydrochloride, pyra factor-1 receptor inhibitor, interferon agonists, interferons, Zoflurin, riboprine, rogletimide, Safingol, Safingol hydrochlo interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, ride, Semustine, SimtraZene, Sparfosate Sodium, SparSomy irinotecan, iroplact, irSogladine, isobengaZole, isohomohali cin, Spirogermanium hydrochloride, Spiromustine, condrin B, itasetron, jasplakinolide, kahalalide F, lamel Spiroplatin, Streptonigrin, Streptozocin, Sulofenur, tallisomy larin-N triacetate, lanreotide, leinamycin, lenograstim, len cin, taxol, tecogalan Sodium, tegaflir, teloxantrone hydro tinan Sulfate, leptolstatin, letrozole, leukemia inhibiting chloride, temoporfin, tenipoSide, teroxirone, teStolactone, factor, leukocyte alpha interferon, leuprolide--estrogen thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, progesterone, leuprorelin, leVamisole, liarozole, linear topotecan hydrochloride, toremifene citrate, treStolone polyamine analogue, lipophilic disaccharide peptide, lipo acetate, triciribine phosphate, trimetrexate, trimetrexate glu philic platinum compounds, lisSoclinamide 7, lobaplatin, curonate, triptorelin, tubulozole hydrochloride, uracil mus lombricine, lometrexol, lonidamine, loSOXantrone, lovasta US 2003/0072794 A1 Apr. 17, 2003 tin, loxoribine, lurtotecan, lutetium teXaphyrin, lySofylline, nimex, urogenital Sinus-derived growth inhibitory factor, lytic peptides, maitansine, mannostatin A, marimastat, urokinase receptor antagonists, Vapreotide, Variolin B, masoprocol, maspin, matrily Sin inhibitors, matrix metallo VelareSol, Veramine, Verdins, Verteporfin, Vinorelbine, VinX proteinase inhibitors, menogaril, merbarone, meterelin, altine, Vitaxin, Vorozole, Zanoterone, Zeniplatin, Zilascorb, methioninase, metoclopramide, MIF inhibitor, mifepristone, Zinostatin Stimalamer. miltefosine, mirimostim, mismatched double stranded RNA, 0158 pH-Sensitive Peptide-DNA Complexes mitoguaZone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-Saporin, mitoxantrone, 0159. In a further embodiment of the invention, the genes mofarotene, molgramoStim, monoclonal antibody, human in plasmid DNA are brought in interaction with fusogenic chorionic gonadotrophin, monophosphoryllipid A+myobac peptide/NLS conjugates. In a further embodiment the NLS terium cell wall Sk, mopidamol, multiple drug resistance moiety is a stretch of histidyl residues able to assume a net gene inhibitor, multiple tumor SuppreSSor 1-based therapy, positive charge at a pH of about 5 to 6 and to show a mustard anticancer agent, mycaperoxide B, mycobacterial reduction or loose completely this charge at pH above 7. The cell wall extract, myriaporone, N-acetyldinaline, N-Substi electroStatic interaction of these positively-charged peptides tuted benzamides, nafarelin, nagreStip, naloxone +pentaZO with the negatively-charged plasmid DNA molecules, estab cine, napavin, naphterpin, nartograstim, medaplatin, nemo lished at pH 5-6 is weakened at physiological pH (pH rubicin, neridronic acid, neutral endopeptidase, nilutamide, Sensitive peptide-DNA complexes). nisamycin, nitric oxide modulators, nitroxide antioxidant, 0160 The first step of the present invention involves nitrullyn, 06-benzylguanine, octreotide, okicenone, oligo complex formation between the plasmid or oligonucleotide nucleotides, onapristone, ondansetron, Ondansetron, oracin, DNA with the histidyl/fusogenic peptide conjugate and lipid oral cytokine inducer, Ormaplatin, OSaterone, Oxaliplatin, components in 10-90% ethanol at pH 5.0 to 6.0. The oXaunomycin, paclitaxel analogues, paclitaxel derivatives, conditions must be where the histidyl residues have a net palauamine, palmitoylrhizoxin, pamidronic acid, panax positive charge and can establish electroStatic interactions ytriol, panomifene, parabactin, paZelliptine, pegaspargase, with plasmids, oligonucleotides or negatively-charged peldesline, pentosan polysulfate Sodium, pentostatin, pentro drugs. At the same time, the presence of the positively Zole, perflubron, perfosfamide, perillyl alcohol, phenazino charged lipid molecules promotes formation of micelles. At mycin, phenylacetate, phosphatase inhibitors, picibanil, the Second step, micelles are converted into liposomes by pilocarpine hydrochloride, pirarubicin, piritrexim, placetin dilution with water and mixing with pre-made liposomes or A, placetin B, plasminogen activator inhibitor, platinum lipids at pH 5-6. This is followed by dialysis against pH 7 compleX, platinum compounds, platinum-triamine complex, and extrusion through membranes, entrapping and encapsu porfimer Sodium, porfiromycin, propyl bis-acridone, proS lating plasmids or oligonucleotides to with a very high yield. taglandin J2, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein 0.161 Whereas the composition of peptides and cationic kinase C inhibitors, microalgal., protein tyrosine phoS lipids in the first step provides the lipids of the internal phatase inhibitors, purine nucleoside phosphorylase inhibi bilayer, the type of liposomes or lipids added at Step 2 tors, purpurins, pyrazoloacridine, pyridoxylated hemoglobin provide the external coating of the final lipoSome formula polyoxyethylene conjugate, raf antagonists, raltitrexed, tion (FIG. 1). Examples for the formulations of peptides ramosetron, ras fameSyl protein transferase inhibitors, ras include: HHHHHSPSL (SEQ ID NO:623), and HHHHH inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhe SPS(LAI) (SEQ ID NO:624). nium Re 186 etidronate, rhizoxin, ribozymes, RII retina 0162 These are added at a 1:0.5:0.5 molar ratio (negative mide, rogletimide, rohitukine, romurtide, roquinimex, charge on DNA: cationic liposome: histidine peptide). The rubiginone B 1, ruboxyl, Safingol, Saintopin, SarCNU, Sar peptide inserts in an alpha-helical conformation inside the cophytol A, SargramoStim, Sdi 1 mimetics, Semustine, Senes lipid bilayer and not only carries out DNA condensation but cence derived inhibitor 1, Sense oligonucleotides, Signal also endowS membrane fusion properties to the complex to transduction inhibitors, Signal transduction modulators, improve entrance across the cell membrane. The type of Single chain antigen binding protein, sizofiran, Sobu ZOxane, hydrophobic amino acids (for example, content in aromatic Sodium borocaptate, Sodium phenylacetate, Solverol, amino acids), in the peptide chain is very important as is the Somatomedin binding protein, Sonermin, Sparfosic acid, length of the peptide chain in ensuring integrity and rigidity Spicamycin D, Spiromustine, Splenopentin, Spongistatin 1, of the complexes. Coating the outer Surface of the com Squalamine, Stem cell inhibitor, Stem-cell division inhibitors, plexes with polyethyleneglycol, hyaluronic acids and other Stipiamide, Stromelysin inhibitors, Sulfinosine, Superactive polymers conjugated to lipids gives the particles long cir vasoactive intestinal peptide antagonist, Suradista, Suramin, culation properties in body fluids and the ability to target Swainsonine, Synthetic glycosaminoglycans, tallimustine, Solid tumors and their metastases after intravenous injection, tamoxifen methiodide, tauromustine, tazaroteine, tecogalan and also the ability to cross the tumor cell membrane. Sodium, tegafur, tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaox 0163 Protease-Sensitive Linkages in Peptides Between ide, tetraZomine, thaliblastine, thalidomide, thiocoraline, the NLS and Fusogenic Moieties thrombopoietin, thrombopoietin mimetic, thymalfasin, thy mopoietin receptor agonist, thymotrinan, thyroid stimulating 0.164 Conversion of Micelles Into Liposomes hormone, tin ethyl etiopurpurin, tirapazamine, titanocene 0.165 An important issue of the present invention is the dichloride, topotecan, topSentin, toremifene, totipotent Stem conversion of micelles formed between the DNA and the cell factor, translation inhibitors, tretinoin, triacetyluridine, cationic lipids, in the presence of ethanol, into lipoSomes. triciribine, trimetrexate, triptorelin, tropisetron, turosteride, This is done by the direct addition of the micelle complex tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ube into an aqueous Solution of preformed liposomes. The US 2003/0072794 A1 Apr. 17, 2003 liposomes have an average Size of 80-160 nm or Vice versa, tification and Selection of regulatory DNA sequences depend leading to a Solution of a final ethanol concentration below on tedious procedures Such as transcription factor footprint 10%. A formulation suitable for pharmaceutical use and for ing in vitro or in Vivo, or Subcloning of Smaller fragments injection into humans and animals will require that the from larger genomic DNA sequences upstream of reporter liposomes are of neutral composition (Such as cholesterol, genes. These methods have been used primarily to identify PE, PC) coated with PEG. regions proximal to the 5' end of genes. However, in many 0166 However, another important aspect is the research instances, regulatory regions are found at considerable dis application of the present invention, Such as for transfection tances from the proximal 5' end of the gene, and confer cell of cells in culture. The composition of the aqueous Solution type- or developmental Stage- Specificity. For example, of liposomes is any type of liposomes containing cationic lipids and suitable therefore for transfection of cells in Studies from the groups of Grosveld and Engel (Lakshmanan culture such as DDAB:DOPE 1: 1. These liposomes are et al., 1999) have shown that over 625 kb of genomic preformed and downsized by Sonication or extrusion Sequences Surrounding the GATA-3 locus are required for through membranes to a diameter of 80-160 nm. The etha the correct developmental expression of the gene in trans nolic micelle preparations are then added to the aqueous genic mice. Extensive DNA stretches at distances 5-20 kb Solution of liposomes with a concomitant dilution of the upstream of the gene were found to be responsible for the ethanol solution to below 10%. This step will result in central nervous System-specificity of expression. The region further condensation of DNA or interaction of the nega between 20 to 130 kb upstream of the gene harbored tively-charged phosphate groups on DNA with positively regulatory regions for urogenital-specific expression of charged groups on lipids. Care must be taken So as only part GATA-3, whereas sequences 90-180 kb downstream of the of the negative charges on DNA are neutralized by lipids in gene conferred endocardial-specific expression. the micelle. The remaining charge neutralization of the DNA is to be provided by the cationic component of the preformed 0171 The presently disclosed method has the potential of liposomes in the Second step. rapidly identifying regulatory control regions. In cells, chro 0167 Regulatory DNA and Nuclear Matrix-Attached matin loops are formed and different attachment regions are DNA used in different cell types or stages of development to 0.168. In a further embodiment of the present invention, modulate the expression of a gene. The presently disclosed the genes in plasmid DNA are driven by regulatory DNA method for isolating regulatory regions based on their Sequences isolated from nuclear matrix-attached DNA using attachment to the nuclear matrix can identify regulatory shotgun Selection approaches. regions irrespective of their distance from the gene. 0169. The compact structural organization of chromatin Although the human genome project is expected to be and the proper Spatial orientation of individual chromo almost complete by the year 2000, information on the Somes within a cell are partially provided by the nuclear location and nature of the vast majority of the estimated matrix. The nuclear matrix is composed of DNA, RNA and 500,000 regulatory regions will not be available. proteins and Serves as the site of DNA replication, gene transcription, DNA repair, and chromosomal attachment in Example 1 the nucleus. Diverse sets of DNA sequences have been found associated with nuclear matrices and is referred to as 0172 Plasmid DNA condenses with various agents, as matrix attachment regions or MARS. The MARS serve many well as various formulations of cationic liposomes. The functions, acting as activators of gene transcription, Silenc condensation affects the level of expression of the reporter ers of gene expression, insulators of transcriptional activity, beta-galactosidase gene after transfection of K562 human nuclear retention Signals and origins of DNA replication. erythroleukemia cell cultures. Liposome compositions are Current studies indicate that different Subsets of MARS are shown in the Table below and in FIG.2. All lipids were from found in different tissue types and may assist in regulating the Specific functions of cells. The presence of this complex Avanti Polar Lipids (700 Industrial Park Drive, Alabaster, asSortment of Structural and regulatory molecules in the Ala. 35007). The optimal ratio of lipid to DNA was 7 matrix, as well as the in Situ localization of DNA replication mmoles total lipid/ug DNA. The transfection reagent (10 ug and transcription complexes to the matrix Strongly Suggest DNA mixed with 70 mmoles total lipid) was transferred to that the nuclear matrix plays a fundamental, unique role in a small culture flask followed by the addition of 10 ml K562 nuclear processes. The Structuring of genomes into domains cell culture (about 2 million cells total); mixing of cells with has a functional significance. The inclusion of Specific MAR the transfection reagent was at 5-10 min after mixing DNA elements within gene transfer vectors could have utility in with liposomes. Cells were assayed for beta-galactosidase many experimental and gene therapy applications. Many activity several times at 1-30 days post-transfection. The gene therapy applications require Specific expression of one transfected cells were maintained in cell culture as normal or more genes in targeted cell types for prolonged time cell cultures. periods. MARS within vectors could enhance transcription of the introduced transgene, prolong the retention of that 0173 Best results were obtained when the cells used for Sequence within the nucleus or insulate expression of that transfection were at low number, not near confluence. In all transgene from the expression of a cotransduced gene experiments the transfection material was added directly in (reviewed by Boulikas, 1995; Bode et al., 1996). the presence of Serum and antibiotics without removal of the 0170 Various biochemical procedures have been used to transfection reagent or Washings of the cells. This simplifies identify regulatory regions within genes. Traditionally, iden the transfection procedure and is Suitable for lymphoid and US 2003/0072794 A1 Apr. 17, 2003 20 other type of cell cultures that do not attach to the dish, but 0.178 At this stage, weak complexation of plasmid DNA grow in Suspension. All DNA condensing agents were with neutral (Zwitterionic) liposomes takes place. This purchased from Sigma. They were Suspended at 0.1 mg/ml ensures homogeneous distribution of plasmid DNA to lipo in water. Plasmid pCMVB was purchased from Clontech and Somes at the Subsequent Step of addition of cationic lipo was purified using the Anaconda kit of Althea Technologies somes. After complexation of plasmid DNA with Zwitteri (San Diego, Calif.). PolyK is polylysine, mw 9,400. PolyR onic liposomes, 50 ul of cationic liposomes (DC-Chol 1 is polyarginine. PolyH is polyhistidine. umole/ml:DOPE 1.4 umole/ml) were added and incubated at 0174) To 100 ul plasmid solution (10 ug total plasmid room temperature for 10 min. At this Stage, a mixed lipo DNA) 20 ul or 50 ul of polyK, polyR, polyH, were added; Some population is present and, most likely, formation of a the volume was adjusted to 250 ul with water followed by type of liposome-DNA complexes containing lipids from the addition of about 70 ul liposomes (7 minoles (ug DNA). After Zwitterionic and cationic lipids takes place. The material was incubation for 10 mm to 1 h at 20° C. the transfection injected (0.35 ml total volume) to the intraperitoneal cavity mixture was brought in contact with the cell culture. The of the animal. At 5 days post-injection the animal was best DNA condensing reagent was polyhistidine compared Sacrificed, the skin was removed and the carcass was incu with the popular polylysine. The best cationic lipid was bated into X-gal staining solution for about 30 min at 37 C. DC-cholesterol (DC-CHOL: The animal was incubated in fixative in X-gal Staining for 0175 3RN-(N',N'-dimethylaminoethane)carbamoyl about 30 min (addition of 100 ul concentrated glutaralde cholesterol). SFV is Semliki Forest virus expressing beta hyde to 30 ml X-gal staining Solution) and the incubation in galactosidase. The results are shown in FIG. 2. Staining Solution continued. Photos were taken in a time

Liposome Molecular weight Composition Preparation L2 DDAB mw 631 DDAB 4.2 umoles/ml 15 mg DDAB + DOPE mw 744 DOPE 4.2 umoles/ml 0.88 ml 20 mg/ml DOPE L3 DOGS-NTA mw 1015.4 DOGS-NTA 1 umole?ml 5 mg DOGS DOPE 1 tumole?ml O.185 m DOPE L4 DC-Chol (mw 537) DC-Chol 1 tumole/ml 0.106 ml DC-Chol (25 DOPE (mw 744) DOPE 1 tumole?ml mg/ml) + 0.185 ml DOPE (20 mg/ml) L5 DOTAP (mw 698) DOTAP 1.4 umole/ml 0.5 ml 10 mg/ ml DOTAP + DOPE (mw 744) DOPE 1.3 tumole?ml 0.25 ml DOPE (20 mg/ml) L6 DODAP (mw 648) DODAP 1.54 umoles/ml 0.5 ml 10 mg/ml DOPE 1.3 tumole?ml DODAP = 5 mg = 7.72 Atmoles + 0.25 ml DOPE (20 mg/ml)

Example 2 course during the incubation period revealing the preferred organs where beta-galactosidase expression took place. 0176 Targeting Genes to Tumors. Using Gene Vehicles (Lipogenes). 0179 Because of the tumor vasculature targeting shown 0177. As shown in FIG. 3, tumor targeting in SCID in FIG. 3E, the data imply that transfer of the genes of (severe combined immunodeficient) mice were implanted angiostatin, endostatin, or oncostatin to the tumors (whose subcutaneously, at two sites, with human MCF-7 breast gene products restrict vascular growth and inhibit blood cancer cells. The cells were allowed to develop into large, Supply to the tumor) is expected to be a rational approach for measurable Solid tumors at about 30 days post-inoculation. Mice were injected intraperitoneously with 0.2 mg plasmid cancer treatment. Also, a combination therapy using anti pCMVB DNA (size of the plasmid is ~4 kb) per animal cancer lipogenes with encapsulated drugs into tumor target carrying the bacterial beta-galactosidase reporter gene. Plas ing liposomes appears as a rational cancer therapy. mid DNA (200 ug, 2.0 mg/ml, 0.1 ml) was incubated for 5 min with 2001 neutral liposomes of the composition 40% 0180. It is to be understood that while the invention has cholesterol, 20% dioleoylphosphatidylethanola been described in conjunction with the above embodiments, mine(DOPE), 12% palmitoyloleoylphosphatidylcholine that the foregoing description and the following examples (POPC), 10% hydrogenated soy phosphatidylcholine are intended to illustrate and not limit the Scope of the (HSPC), 10% distearoylphosphatidylethanolamine (DSPE), invention. Other aspects, advantages and modifications 5% sphingomyelin (SM), and 3% derivatized vesicle-form within the Scope of the invention will be apparent to those ing lipid M-PEG-DSPE. skilled in the art to which the invention pertains. US 2003/0072794 A1 Apr. 17, 2003 21

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 B galactosidase or pyruvate kinase at the gene level and microinjection of plasmids 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 microinjec tion in Xenopus oocytes. The PKKGSKKA from Xenopus H2B was in effective and PKTKRKV was less effective. CGYGPKKKRKVGG (SEQ ID Synthetic peptide from SV40 large T antigen conjugated to NO:23) various 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 mm 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. A mutated (R->I) version of SV40 large T NLS. Effective NLS. Yeast GALA (99 kD). Fusions of the GAL4 gene portion encoding the 74 N-terminal amino acid with E. coli f-galactosidase introduced into yeast cells specify nuclear localization. 74 N-terminal amino acid MKXCRLKKLKCSKEKPKC Yeast GALA. Acted as an efficient nuclear localization sequence A (SEQ ID NO:28) when fused to invertase but not to B-galactosidase introduced 29 N-terminal amino acid by transformation into yeast cells. Polyoma large T protein. Identified PKKARED (SEQ ID NO:29) by fusion with pyruvate kinase cDNA and microinjection of Vero VSRKRPR (SEQ ID NO:30) African green monkey cells. Mutually independent NLS. Can exert cooperative effects. CGYGVSRKRPRPG Polyoma virus large T protein. This synthetic peptide crosslinked (SEQ ID NO:31) O 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 he 5' terminal portion of SV40 VP1 gene and the complete cDNA sequence of poliovirus capsid VP1 replacing the VP1 gene of SV40. 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 f-galactosidase gave a protein that was transported to the nucleus in yeast cells. Subtractive constructs of VP1 lacking A' to C" were cytoplasmic. This, FITC-labeled, synthetic peptide crosslinked o BSA or IgG, caused nuclear import after microinjection into 3T6 cells. Replacement of K with T did not. 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 VP1 capsid protein at the gene level resulted in nuclear import of the hybrid VP1 in simian cells infected with the hybrid SV40. EEDGPQKKKRRL (307–318) Polyoma virus capsid protein VP2. A construct having truncated (SEQ ID NO:35) VP2 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 f-galactosidase. KRPRP (SEQ ID NO:37) Adenovirus E1a. 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 E1a. This synthetic peptide crosslinked NO:38) to chicken bovine albumin and microinjected into HeLa cells caused nuclear localization. LVRKKRKTESP (NLS 1) Xenopus N1 (590 amino acid). Abundant in X. laevis oocytes, (SEQ ID NO:39) forming complexes with histones H3, H4 via two acidic domains LKDKDAKKSKQE (NLS2) each containing 21 and 9 (D + E), respectively. The NLS1 is (SEQ ID NO:40) required but not sufficient for nuclear accumulation of protein N1. NLS 1 and 2 are contiguous at the C-terminus. US 2003/0072794 A1 Apr. 17, 2003 22

TABLE 3-continued Simple NLS Signal oligopeptide Protein and features GNKAKRORST v-Rel or p59'' 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 f-galactosidase directed that protein to the nucleus. PFLDRLRRDOK 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 PKOKRKMAR NS1 in recombinant SV40. The 1st NLS is conserved among all NS1 (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 L. NLS inferred from its sequence similarity to (SEQ ID NO:46) human lamin A NLS. CVRTTKGKRKRIDV Xenopus lamin L. This synthetic peptide crosslinked to chicken (SEQ ID NO:47) bovine albumin and microinjected into HeLa cells caused nuclear localization. ACDKRVKLD Human c-myc oncoprotein. This synthetic peptide crosslinked to (SEQ ID NO:48) chicken bovine albumin and microinjected into HeLa cells caused nuclear localization. ACDKRVKLD Human c-myc oncoprotein. Conjugation of the M1 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. RORRNELKRSP (SEQ ID NO:50) (M2, medium potency NLS) SALIKKKKKMAP Murine c-abl (IV) gene product. The p16088 has a cytoplasmic (SEQ ID NO:51) and plasma membrane localization, whereas the mouse type IV c-abl protein is largely nuclear. 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) NLS1 determined by KIKGIQQATA (497-524 amino fusion with B-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. RKDRRGGRMLKHKRORDD Human ER (estrogen receptor, 595 amino acid) NLS. NLS is between GEGRGEVGSAGDMRAMINO the hormone-binding and DNA-binding regions; ER, in contrast with ACIDNLWPSPLMIKRSKK GR, lacks a second NLS. Can direct a fusion product with B (amino acid 256-303) galactosidase to the nucleus. (SEQ ID NO:55) RKFKLKFNK 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 Ets1 core NLS. Within a 77 amino acid C-terminal segment 90% homologous to Ets2. When deleted by deletion mutagenesis at the gene level the mutant Ets1 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 E1a. 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 off-galactosidase KHRRHP (SEQ ID NO:68) fusion protein. KHKKHP (SEQ ID NO:69) US 2003/0072794 A1 Apr. 17, 2003 23

TABLE 3-continued Simple NLS

Signal oligopeptide Protein and features RHLKHP (SEQ ID NO:70) KHRKYP (SEQ ID NO:71) KHRQHP (SEQ ID NO:72) PETTVVRRRGRSPRRRTPSP Double NLS of hepatitis B virus core antigen. The two underlined RRRRSPRRRRSQS (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. 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 Mx1 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) CGYGARKTKKKK (human glucocorticoid receptor) (SEQ ID NO:79) CGYGIRKDRRGGR (human estrogen receptor) (SEQ ID NO:80) CGYGARKLKKLGN (human androgen receptor) (SEQ ID NO:81) RKRORALMLROAR 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 (T,) plasmid. A fusion protein with f (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, N'-dimethylguanosine-specific tRNA methyltransferase. Localizes at the nuclear periphery. The 70-213 amino acid segment of TRM1 causes nuclear localization off galactosidase 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. US 2003/0072794 A1 Apr. 17, 2003 24

0181)

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 TKKAGOAKKKKLD 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. CGOAKKKKLD Xenopus nucleoplasmin-derived synthetic peptide; crossliniked 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. HRKYEAPRHxPRKR (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 f3-galactosidase were used to transform yeast cells followed by fluorescence staining with b-gal antibody. The 373 amino acid of L3 fused to B-gal failed to localize to the nucleus, unless a 8 amino acid bridge containing a proline was inserted between L3 and f-gal. NKKKRKLSRGSSOKTKGTSASAK SV40 Vp3 structural protein. (35 amino acid C-terminus). By ARHKRRNRSSRS (one sequence) DEAE-dextran-mediated transfection of TC7 cells with mutated (SEQ ID NO:93) constructS. RVTIRTWRVRRPPKGKHRK Simian sarcoma virus v-sis gene product (p28). 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 ammo 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. KRKEEPEPEPKKAK 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-blastula gastrula 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 of anaphase/G1 due to dephosphorylation of NLS. NLS confers cell cycle-regulated nuclear import of SWI5-f-galactosidase fusion protein. 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. 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(ADP ribosyl)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 US 2003/0072794 A1 Apr. 17, 2003 25

TABLE 4-continued

& Bipartite or "split' NLS Signal Oligopeptide Protein and features 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 f glucuronidase 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.

0182

TABLE 5 Nonpositive NLS' lacking clusters of arginines/lysines Signal oligopeptide Protein and features OLVWMACNSAMINO Influenza virus nucleoprotein (NP). The underlined region ACDFEDLRVLSFIRGTKVS (327–345) when fused to chimpanzee a-globin at the cDNA level and PRG 327-356 microinjected into Xenopus oocytes specifies nuclear localization. (SEQ ID NO:105) MNKIPIKDLLNPO Yeast MAT a2 repressor protein, containing a homeodomain. (NLS 1 at N-terminus) (SEQ ID The two NLS are distinct, each capable of targeting f-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,Kx. KIPRXHFY Drosophila HP1 (206 amino acids) that binds to EERLSWYSDNED (SEQ ID heterochromatin and is involved in gene silencing. NLS identified by B NO:108) galactosidase/HP1 fusion proteins introduced by P-element mediated 152-206 (C-terminal transformation into Drosophila embryos. segment) Adenovirus type 5 E1A internal, developmentally-regulated MxSLXYMxMF NLS. This NLS functions in Xenopus oocytes but not in somatic cells. This NLS can be utilized up to the early neurula stage.

0183)

TABLE 6 Nucleolar localization signals (NoLS Signal oligopeptide Protein and features

MPKTRRRPRRSORKRPPTP Nucleolus localization signal in amino terminus of human p27*. (SEQ ID NO:109) "protein (also called Rex) of T cell leukemia virus type I (HTLV-I). When this peptide is fused to N-terminus of B galactosidase, 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 ?-galactosidase was functional in nuclear targeting. US 2003/0072794 A1 Apr. 17, 2003 26

TABLE 6-continued Nucleolar localization signals (NoLS Signal oligopeptide Protein and features GRKKRRORRRP 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 1). The 3 amino acids shown in (SEQ ID NO:112) parenthesis are essential for the localization of the f galactosidase to the nucleolus. The 9 amino acid basic region is able to localize f-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. FKRKHKKDISONKRAVRR 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 heat shock. 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. 3-galactosidase fused to RW was targeted to the nucleus, and fused to the entire 35-50 region, was targeted to the nucleolus. RQARRNRRRRWRERORO (35-51) HIV-1 Rev protein. A fusion of this Rev peptide with f3– (SEQ ID NO:116) galactosidase became nuclear but not nucleolar. The 1-59 amino acid segment of Rev fused to f-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.

0184

TABLE 7 Karyophilic clusters on non-membrane protein kinases Non-membrane Karyophilic peptides protein kinase Species Features 73 FWVHKRCHE Protein kinase C (673 Bovine, human Known to translocate to the (SEQ ID NO:117) aa) f type nucleus following treatment of 96 DDPRSKHKFKIH cells with mitogens. (SEQ ID NO:118) (SEQ ID NO:119) 71 FVVHRRCHIEF Protein kinase C (697 bovine, human Y (SEQ ID NO:120) aa) type 95 DDPRNKHKFRLH (SEQ ID NO:121) 591 TKHPAKRLG (SEQ ID NO:122) 72 FVVHKRCHE Protein kinase C (673 rabbit type C. and (SEQ ID NO:123) aa) B 96 DDPRSKHKFKIH (SEQ ID NO:124) (SEQ ID NO:125) 71 FVVHRRCHE PKC-I (701 aa) rat brain (SEQ ID NO:126) 95 DDPRNKHKFRLH (SEQ ID NO:127) (SEQ ID NO:128) 22 GENKMKSRLRKG Protein kinase C Drosophila 14 exons, 20 kb; 3 transcripts in (not conserved) (639 aa, 75 kDa) adult flies; not expressed in 0–3 h (SEQ ID NO:129) Drosophila embryos; the 8OSYWVHKRCHEYVT VVHKRCHE (SEQ ID (conserved) NO: 133)motif (or VVHRRCHE US 2003/0072794 A1 Apr. 17, 2003 27

TABLE 7-continued Karyophilic clusters on non-membrane protein kinases Non-membrane Karyophilic peptides protein kinase Species Features (SEQ ID NO:130) (SEQ ID NO:134)) is conserved 211 PDDKDOSKKKTR among all PKC known. TIK (not conserved) (SEQ ID NO:131) 614PPFKPKIKHRKMC P (not conserved) (SEQ ID NO:132) 148 KKVLODKRFK Glycogen synthase rat brain Phosphorylates glycogen synthase, NRELQIMRKLD (SEQ kinase 3 c-Jun, c-Myb; two isoforms ID NO:135) GSK-3C encoded by discrete genes; highly (483 aa) expressed in brain; both C. and f forms are cytosolic but also associated with the plasma membrane consistent with their role in signal transduction from the cell surface. LODRRFKNRELO Zw3 Drosophila Product of the segment polarity (SEQ ID NO:136) 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 each segment. 289ECLKKFNARRKL Ca/calmodulin- rat brain Composed of nine 50 kDa C KGAIL dependent protein subunits and three 60 kDa B (SEQ ID NO:137) kinase II (CaM kinase subunits; both are catalytic; II) f subunit (542 aa, calmodulim- and ATP-binding 60.3 kDa) domains; highly expressed in forebrain neurons, concentrated in postsynaptic densities; acts as a Ca'-triggered switch and could be involved in long-lasting changes in synapses. 29OLKKFNARRKL CaM kinase II (478 rat brain This particular isoform is KGAILTTM (SEQ ID aa, 54 kDa) exclusively expressed in the brain; NO:138) C-subunit high enzyme levels in specific 45OEETRVWHRRDGK 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 (R) and two catalytic (C) subunits; cAMP releases the C subunit from the inactive R.C. 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 EEEIOELKRKLH CGDPK (SEQ ID bovine lung By protein sequencing; composed KCOSVLP (SEQ ID NO: 144) of two identical subunits activated NO:142) (670 aa, 76.3 kDa) in an allosteric manner by binding 389 KLKKRHIVDTR of c(3MP and not by dissociation (SEQ ID NO:143) of catalytic subunit as in cADPK: sequence similar to cADPK 117 KTLKKHTVK 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. US 2003/0072794 A1 Apr. 17, 2003 28

TABLE 7-continued Karyophilic clusters on non-membrane protein kinases Non-membrane Karyophilic peptides protein kinase Species Features 16SHGHG SNF1 (633 aa, 72 kDa) S. cerevisiae Serf Thr kinase; 166 EYCHRHKVHRD 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 cAMP dependent protein kinase (cADPK). 7OPVKKKKKREIK Casein kinase II (Cl- Drosophila CKIL is composed of a and 3 (SEQ ID NO:148) subunit, catalytic) nelanogaster subunits in a a2f32 130-150 kDa 269 DILORHSRKRW (336 aa) protein; the a-subunit is the ERF (SEQ ID NO:149) catalytic and the f3is 146 PKSSRHHHTDG CKII (B-subunit, Drosophila autophosphorylated. (SEQ ID NO:150) regulatory) (215 aa) melanogaster 142 PKSSRHHHTDG CKII (B-subunit, bovine (lung) (SEQ ID NO:151) regulatory) (209 aa, 24.2 kDa) 108 PKORHRKSLG KIN1 (1064 aa, 117 S. cereviszae 30% aa similarity to bovine (SEQ ID NO:152) kDa) cADPK and 27% (KINI) or 25% 129 GSMCKVKLAK (KJN2) aa similarity to v-Src HRYTNE within the kinase domain; the (SEQ ID NO:153) catalytic domains of KINi and 506 DRKHAKIRNO KIN2 are near the N-terminus and (SEQ ID NO:154) are structural mosaics with features 638 GNIFRKLSORR characteristic of both Tyr and KKTIEO Serf Ihr kinases. (SEQ ID NO:155) 773 PPLNVAKGRKL HP (SEQ ID NO:156) 87 ELROFHRRSLG KIN2 (1152 aa, 126 S. cerevisiae (SEQ ID NO:157) kDa) 111 GKVKLVKHRO TKE (SEQ ID NO:158) 217 GSLKEHHARKF ARG (SEQ ID NO:159) 807 LSVPKGRKLHP (SEQ ID NO:160) 6OFLRRGIKKKLTLD STE7 (515 aa) S. cerevisiae Implicated in the control of the (SEQ ID NO:161) three cell types in yeast: (a,a,and 472 PSKDDKFRHWC ala) of which a and a cells are RKKSKKEDKRKRE haploid and are specialized for (SEQ ID NO:162) mating whereas afa cells are diploid and are specialized for meiosis and sporulation; with the exception of the mating type locus, MAT, all cells contain the same DNA sequences. STE7 gene produces insensitivity to cell division arrest induced by the yeast mating hormone, a-factor. 722 ORRVKKLPSTTL S6KIIC (733 aa) Xenopus (SEQ ID NO:163) ORRVKKLPSITL S6KII B Xenopus (SEQ ID NO:164) 742 ORRVKKLPSTTL S6KII (752 aa) Chicken (SEQ ID NO:165) 713ORRVRKLPSTTL S6KII (724 aa) Mouse (SEQ ID NO:166) 16GVVYKGRHKTTG CDC2HS Human Isolated by expressing a human (SEQ ID NO:167) (297 aa) cDNA library in S. pombe and 12O FCHSRRVLHRD selecting for clones that LKP (SEQ ID NO:168) complement a mutation in the cdc2 yeast gene; the human CDC2 gene can complement both the inviability of a null allele of S. cerevisiae CDC28 and cdc2 mutants of S. pombe; CDC2 mRNA appears after that of CDK2. US 2003/0072794 A1 Apr. 17, 2003 29

TABLE 7-continued Karyophilic clusters on non-membrane protein kinases Non-membrane Karyophilic peptides protein kinase Species Features GVVYKARHKLSGR cdc2 (297aa) S. pombe High homology to S. cerevisiae (SEQ ID NO:169) CDC28. 119SHRVLHRDLKP CDK2 (cell division Human The human CDK2 protein has 65% (SEQ ID NO:170) kinase 2) (298 aa) sequence identity to human p34° and 89% sequence identity to Xenopus Eg1 kinase; human CDK2 was able to complement the inviability of a null allele of S. cerevisiae CDC28 but not calc2 mutants in S. pombe. CDK2 mRNA appears in late G1/early S. O9 FCHSHRVLHRD Eg1 (297 aa) Xenopus Cdk2-related LKP (SEQ ID NO:171) 25 GAYCHSHRILH CDC28 (298 a) S. cerevisiae The homolog of S. pombe Cdc2 RDLKP (SEQ ID NO:172) 19 HSHRVIHRDLKP cdk3 (305 aa) Human (SEQ ID NO:173) 56 KELKHKNVR PSSALRE (291 aa) Human cdc2-related kinase. (SEQ ID NO:174) (SEQ ID NO:175) MDRMKKIKRQ (N- PCTAIRE-1 (496 aa) Human cdc2-related kinase. erminus) (SEQ ID NO:176) 41 DKPLSRRLRRV (SEQ ID NO:177) MKKFKRR PCTAIRE-2 (523 aa) Human cdc2 related kinase. (SEQ ID NO:178) 29 RNRHRRIS (SEQ ID NO:179) 72 SRRSRRAS (SEQ ID NO:180) (SEQ ID NO:181) 512 GHGKNRROSM LF (SEQ ID NO:182) 163 HTRKLHR PCTAIRE-3 Human cdc2 related kinase. (SEQ ID NO:183) (380 aa) 369 PGRGKNRROSIF (SEQ ID NO:184) 69 EVFRRKRRLH KKIALRE (358 aa) Human cdc2-related kinase. (SEQ ID NO:185) (SEQ ID NO:187) 3O2 DKPTRKTLRKSR KHH (SEQ ID NO:186) 1 MVKRHKNT nim1 gene product S. pombe (SEQ ID NO:188) (new inducer of 87 DGELFHYRKHGP mitosis); protein (SEQ ID NO: 189) kinase (370 aa) 114 DAVAHCHRFRFR HRD (SEQ ID NO:190) 295 KKSSSKKWVRRL OORDD (SEQ ID NO:191) 194 PAOKLRKKNNFD Wee1 gene product S. pombe The Wee1 gene functions as a (SEQ ID NO:192) (877 aa) dose-dependent inhibitor that 388 KOHRPRKNTNFT delays the initiation of mitosis PLPP (SEQ ID NO.193) until the yeast cell has attained a 592 KYAVKKLKVKF certain size; Wee1 has a protein SGP (SEQ ID NO:194) kinase consensus probably regulating cdc2 kinase. 266 PNETRRIKRAN CDC7 (497 aa) S. cerevisiae Required for mitotic but not RAG (SEQ IDNO: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. US 2003/0072794 A1 Apr. 17, 2003 30

TABLE 7-continued Karyophilic clusters on non-membrane protein kinases Non-membrane Karyophilic peptides protein kinase Species Features 48YDHVRKTRVAIKK ERK1 (MAP kinase) Rat Known to translocate to the (SEQ ID NO:196) (367 aa; 42 kDa) nucleus following their activation by phosphorylation at T-190, and Y-192 (T-183, Y-185 in ERK2). 59 ILKHFKHE FUS3 (353 aa) S. cerevisiae MAP-(ERK1)-related. (SEQ ID NO:197) 252 QIKSKRAKEY KSS1 (368 aa) S. cerevisiae MAP-(ERK1)-related. (SEQ ID NO:198) ELVKHLVKHGSN SW16 S. cerevisiae Activator of CACGA-box with (SEQ ID NO:199) (803aa, 90 kDa) sequence similarity to cdc10; GKAKKIRSOLL required at START of cell cycle. (SEQ ID NO:200) EORIKRHRJDVSDED cdc10 S. pombe (SEQ ID NO:201) SNIKSKCRRVW (SEQ ID NO:202) 37 PPKRRTD CTD kinase (528 aa) S. cerevisiae Consists of 3 subunits of 58, 38, (suggested by the 58 kDa subunit and 32 kDa; disruption of the 58 authors) (SEQ ID (catalytic) kDa gene gives cells that lack CTD NO:203) kinase, grow slowly, are cold 492 KLARKOKRP sensitive, but have different (SEQ ID NO:204) phosphorylated forms of RNA pol II. 29 GVSSVVRRCHKP Phosphorylase kinase Rabbit (skeletal (SEQ ID NO:205) (catalytic subunit) muscle) (386 aa) 489 KKYMARRKW Myosin light chain Chicken gizzard Ca'/calmodulin-activated; OKTGHAV 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. RCNRRLKSO kinase (partial 368 muscle) (SEQ ID NO:207) carboxy-terminal aa 334 ILLKKYLMKRR sequence) WKKNFAVS (SEQ ID NO:208) 28 GVSSVVRRCHKP 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 C., f, Y and 8 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.

0185

TABLE 8 Nuclear localization signals on DNA repair proteins

Gene Equivalent protein Putative NLS product in other species Features

HIGHER EUKARYOTES

None ERCC1 RAD10 297 aa; DBD: interacts (N-terminus) strongly with ERCC4 (XPF) MDPGKDKEGvpaps.gppaRKKF to form an excision US 2003/0072794 A1 Apr. 17, 2003 31

TABLE 8-continued Nuclear localization signals on DNA repair proteins Gene Equivalent protein Putative NLS product in other species Features (bipartite NLS) endonuclease; unless the (SEQ ID NO:210) KDKXRKK is a bipartite NLS it may depend upon its binding with ERCC4 for its nuclear import. None ERCC2 RAD3 (S. cer) 760 aa; DNA helicase 681DKRFARGDKRGKLPR (XPD) component of TFIIH, (near the C-terminus) (four essential for cell viability; positive, one negative over a contains one nucleotide heptapeptide stretch) binding, one DNA-binding, (SEQ ID NO:211) and seven domains characteristic of helicases; 52% identity with S. cer RAD3 at the amino acid level. 8 DRDKKKSRKRHYEDEE ERCC3 SSL2 (S. cer) 782 aa; helicase, component (SEQ ID NO:212) (XPB) Haywire(Dros) of TFIIH essential for cell 522 YVAKTKKRILLYTM viability; helix-turn-helix, (SEQ ID NO:213) DNA-BD, and helicase (weak NLS if at all, hydrophobic domains environment) 769 PSKHVHPLFKRFRK (SEQ ID NO:214) 84 KKOTLVKRRORKD ERCC5 RAD2: 1186 aa in human, 1196 in X. (SEQ ID NO:215) (XPG) Rad13 laevis; 3' incision 21O EFTKRRRTL endonuclease; involved in (SEQ ID NO:216) homologous recombination; 390 DESMIKDRKDRLP strongly nuclear (SEQ ID NO:217) 117O GKKRRKLRRARGRK RKT (SEQ ID NO:218) 253POKOEKKPRKIMLNEASG ERCC6 RAD26 1493 aa; involved in the (SEQ ID NO:219) CS-B preferential repair of active 314 PNKKARVLSKKEERLKK genes; nonessential for cell HIKKLQKR (SEQ ID NO:220) viability 406 PLPKGGKROKKVP (SEQ ID NO:221) 455 DGDEDYYKORLRRWNK LRLODKEKRLKLEDDSEESD (SEQ ID NO:222) 1028 DVOTPKCHLKRRIOP XPKRKKFP (SEQ ID NO:223) 1180 KHKSKTKHHSVAEEETL EKHLRPKQKPKX PHLVKK RRY (SEQ ID NO:224) 1324 PAGKKSRFGKKRN (SEQ ID NO:225) 21 PASVRASIERKRORALM XPA RAD14 273 aa; Zinc finger domain; LRGAR (SEQ ID NO:226) involved in lesion 160 PPLKFIVKKNPHHSOW recognition GD (weak) (SEQ ID NO:227) 210 NREKMKOKKFDKKVKE (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) hydrophilic protein; might be 26.2 PSAKGKRNKGGRKKRSK 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:231) genome whereas inactive and 368 RTHRGSHRKDP (weak) the nontranscribed strand of (SEQ ID NO:232) active genes are not repaired 384 SSSSSSSKRGKKMCSDG (SEQ ID NO:233) 531 ALKRHLLKYE (weak) (SEQ ID NO:234) 594 SNRARKARLAEP (SEQ ID NO:235) 660 PNLHRVARKLD (weak) (SEQ ID NO:236) US 2003/0072794 A1 Apr. 17, 2003 32

TABLE 8-continued Nuclear localization signals on DNA repair proteins Gene Equivalent protein Putative NLS product in other species Features 716 ERKEKEKKEKR (SEQ ID NO:237) 740 IRERLKRRYG (SEQ ID NO:238) 8O1 GGPKKTKRERK (SEQ ID NO:239) 2O KSKAKSKARREEEEED XPC 940 aa; the first 117 aa are (SEQ ID NO:240) acking in the Legerski and 54 GKRKRG (SEQ ID NO:241) Peterson, (1992) XPC 69 GPAKKKVAKVTVK sequence (see above); the (SEQ ID NO:242) ollowing 823 aa are 103 PSDLKKAHHLKRG identical. (SEQ ID NO:243) 82 EIDRRKKRPLENDGPVKK Swi4 (S. pom) 1137 aa; mismatch repair KVKKVQQKE (SEQ ID protein; Rep-3 is in the NO:244) immediate 5' flanking region 375 KENVRDKKKG of DHFR gene (89 bp) but (SEQ ID NO:245) ranscribed from the opposite 571 FGRRKLKKWVT strand; a bidirectional (SEQ ID NO:246) promoter is used for both 71OPLIKKRKDEIOG ranscripts. (SEQ ID NO:247) 1091 KELEGLINTKRKRLKYF AKLW (SEQ ID NO:248) 422 EKHEGKHQKLL (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 gene on chromosome 2p16. 397 PDRRLTKKLNKRG MSH2 (SEQ ID NO:250) (S cer) 547 DAKELRKHKKYE (SEQ ID NO:251) 869 VKMAKRKANE (SEQ ID NO:252) 95 GELAKRSERRAEAE Human Rad2 Rad2 (S. pom) 400 aa; required for fidelity (SEQ ID NO:253) of chromosome separation at 354 KRKEPEPKGSTKKKAK mitosis; limited similarity to TG (SEQ ID NO:254) RAD2 (ssDNA nuclease), 394 GKFKRGK (SEQ ID rad 13, and XPG (ERCC5). NO;255) None OSC 339 aa; recombination-repair RAD51 protein: 83% homology to S cerevisiae RAD51 and 55% homology to E. coil RecA. None RAD23 Subunit of XPC (125 kDa) None HHR23A RAD23 Subunit of XPC (125 kDa) 32 PSOAEKKSRARAO 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. GAKKRKDDA ATPase Q1 RecQ (E. coli) 649 aa: altered in XPC cells; (SEQ ID NO:257) undetermined role in repair PKKPRGKM (SEQ ID NO:258) Calf thymus HMG 1 EHKKKHP (SEQ ID NO:259) (259 aa); involved in the ETKKKFKDP (SEQ ID NO:260) recognition of cisplatin EKSKKKK(EID) (SEQ ID lesions NO:261) E.G.KKKKKFAK (SEQ ID NO:262) 512 RDEKKRKOLKKAKAK 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, US 2003/0072794 A1 Apr. 17, 2003 33

TABLE 8-continued Nuclear localization signals on DNA repair proteins Gene Equivalent protein Putative NLS product in other species Features MTFII, LEF-1, TCF-1a, and ABF2. 1 MPKRGKKG (SEQ ID Ref-1 Redox factor 1 from HeLa NO:266) (HAP1) cells; 37 kDa, 318 aa; apurinicfapyrimidinic (AP) endonuclease for DNA repair but also of redox activity stimulating Jun/Fos DNA binding. 1. MPKRGKKG HAP1 ExOIII 323 aa; apurinicfapyrimidinic (SEQ ID NO:267) (bovine) (E. coli) (AP)-endonuclease ExOA (S. pneumoniae) DROSOPHILA 1 MGPPKKSRKDRSGGDKF Haywire ERCC3 (XPB) helicase with 66% identity to GKKRRGODE human ERCC3; flies (SEQ ID NO:268) SSL2 (S. cer) expressing marginal levels of EMSYSRKRORFLVNOG Haywire display motor (weak) (SEQ ID NO:269) defects and reduced life span YYEHRKKNIGSVHPLFK KFRG (bipartite) (SEQ ID NO:270) 77 ARGKKKQPK (SEQ ID Rrp1 HAP1 Recombination repair protein NO:271) 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) 3.36 EEEAKPSTETKPAKGR KKAP (SEQ ID NO:278) 372 KPARGRKKA (SEQ ID NO:279) 394 GSKTTKKAKKAE (SEQ ID NO:280) S. CEREVISIAE 2OO EKLRRKLYISGG RAD1 ERCC4 1100 aa; 30% sequence (SEQ ID NO:281) (XPF) identity to Rad16; RAD1 515 NKKRGVRQVLLN (SEQ Rad16 interacts strongly with ID NO:282) RAD10 565 KEOVTTKRRRTRG (conserved in Rad16) (SEQ ID NO:283) 1024 NLRKKIKSFNKLO (SEQ ID NO:284) 89 RORKERROGKRE RAD2 XPGC 1031 aa, 117.8 kDa, ssDNA (SEQ ID NO:285) Rad13 endonuclease; rad mutants 907 ENKFEKDLRKKLVNNE are defective in incision (SEQ ID NO:286) 984 RDVNKRKKKGKOKRI (SEQ ID NO:287) 101.7 KRISTATGKLKKRXM (SEQ ID NO:288) 672 GKDDYGVMVLADRRF RAD3 ERCC2 or XPD; 778 aa, 89,779 Da; 30% SRKRSQLP (contains the bulky (S. cer) Rad15 or Rhp3 sequence identity to rad16; F) (SEQ ID NO:289) ATP-dependent DNA helicase; single-stranded DNA-dependent ATPase. 26 PLSRRRRVRRKNOPLPD RAD4 XPC 754 aa; mutations in RAD4 AKKKFKTG (SEQ ID NO:290) that that inactivate the 134 NEERKRRKYFHMLYL excision repair function of (SEQ ID NO:291) RAD4 result in truncated 160 EWINSKRLSRKLSNL proteins missing the C (weak) (SEQ ID NO:292) terminal one-third of RAD4. US 2003/0072794 A1 Apr. 17, 2003 34

TABLE 8-continued Nuclear localization signals on DNA repair proteins Gene Equivalent protein Putative NLS product in other species Features 254 EMSANNKRKFKTLKRSD weak (SEQ ID NO:293) 382 WMNSKVRKRRTKDDF GEK (SEQ ID NO:294) 403 RKVITALHHRKRTKID DYED (SEQ ID NO:295) 504 KTGSRCKKVIKRTVGRP (SEQ ID NO:296) 150 FHPKRRRIYGFR (SEQ ID RAD5 1169 aa; helicase involved in NO:297) postreplication-repair (RAD6 215 DSRGRKKASM (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 DEDERRKRRIEE poly (GT) repeats in the yeast (SEQ ID NO:300) genome. 1 MSTPARRRLMRDFKRM RAD6 RAD6 mediates the KEDAPP (SEQ ID NO:301) ubiquitination of H2A and H2B histones 15 GVAKLRKEKSGAD RAD10 ERCC1 210 aa; forms an (SEQ ID NO:302) endonuclease with RAD1; 76 DDYNRKRPFRSTRPGK the basic and tyrosine-rich (SEQ ID NO:303) central domain was suggested to bind DNA by ionic interactions and tyrosine intercalation. 172 EGKAHRREKKYE RAD14 XPAC 247 aa, 29.3 kDa; two zinc (SEQ ID NO:304) fingers; involved in lesion 2OO NRLREKKHGKAHHH recognition; 27% sequence (SEQ ID NO:305) identity and 54% sequence similarity (if conserved residues are grouped together) to human XPA: deletion of RAD14 gene generates high UV sensitivity. 345 ERRKOLKKOGPKRP Ixr1 591 aa; two consecutive (SEQ ID NO:306) (S. 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 483 LTCKKLKTHNRLSG RAD26 ERCC6 1075 aa; disruption of the weak (SEQ ID NO:308) (yeast CS-B (hum) RAD26 gene gives viable 934 NALRKSRKKITKOYEIGT ERCC6) yeast cells unable to PXGEIRKRDP 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 MRE11 Rad32 (S. pom) meiotic recombination (SEQ ID NO:310) protein; functions in the 408 FYKKRSPVTRSKKSG same pathway with RAD51 (SEQ ID NO:311) none; RAD51 RecA (E. coli) 402 aa; essential for repair of 361 GFKKGKGCOR DSBs and recombination; (SEQ ID NO:312) associates strongly with RAD52; self associates: neither RAD51 nor RAD52 possess a typical simple NLS. none; RAD51 (K. 364 aa 328 GFKKGKGCOR lactis) (SEQ ID NO:313) none; RAD52 Rad22 504 aa; rad52 mutants are 155 ERAKKSAVTDALKRSLR defective in ionizing GFGXDKDFLAKIDKVKFDP radiation, mitotic PD (tripartite) recombination, mating-type US 2003/0072794 A1 Apr. 17, 2003 35

TABLE 8-continued Nuclear localization signals on DNA repair proteins Gene Equivalent protein Putative NLS product in other species Features (SEQ ID NO:314) switching, and repair of DSDS 1 MARRRLPDRPP RAD54 898 aa; recombination-repair (SEQ ID NO:315) protein; ATP-binding motif: 65 GGRSLRKRSA helicase domains; in the (SEQ ID NO:316) same subfamily of helicases 99 OLTKRRKD with MOT1 and SNF2. (SEQ ID NO:317) 269 DETVFVKSKRVKASSS RADSS Similarity to RecA, and (extremely weak if at all NLS) lower similarity to RAD51, (SEQ ID NO:318) RAD57, and DMC1 317 GEDRKREGRNLKR (SEQ ID NO:319) 371 PISROSKKRKFDYRVP RAD57 460 aa; nucleotide-binding (SEQ ID NO:320) domain; limited similarity to RAD51 62 GLKKPRKKTKSSRH SSL2 ERCC3 (XPB) 843 aa; putative helicase that (SEQ ID NO:321) seems to function in repair 688 GRILRAKRRNDEG but also in the removal of (SEQ ID NO:322) secondary structures in the 5' 784 GRGSNGHKRFKS (weak) untranslated region of mRNA (SEQ ID NO:323) to allow ribosome binding and scanning. 50 TRRHLCKIKGLSE (weak) DMC1 RecA 334 aa; yeast homolog of (SEQ ID NO:324) RecA, meiosis-specific; 277 DGRKPIGGHXRKGRG dmc 1 mutants are defective DER (bipartite) (SEQ ID in reciprocal recombmation NO:325) and accumulate DSBs 11 ETEKRCKOKEORY PMS 1 904 aa, 103 kDa; mismatch (SEQ ID NO:326) repair protein; MutL (Salmonella) and HexB (Streptococcus) homolog None HRR25 Hhp1, Hhp1 (S. pom) Mutations in HRR25 Ser?hir 1 MDLRVGRKFRIGRKG CR1 (mamm protein kinase cause defects (SEQ ID NO:327) in DNA repair and 139 GRRGXGLSKKYRDFNT retardation in cell cycling HRHIP (Bipartite weak NLS) (SEQ ID NO:328) 96 HELTKRSSRRVETEK 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. 2OO MLAMARRKKKMSAK MOT1 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 LHPEKKRSISE (weak expression of several., but NLS) (SEQ ID NO:332) not all, pheromone responsive genes rn the absence of STEI2; the 1257 to 1825 aa domain (568 aa residues) has homology to SNF2 and RAD54 S. POMBE 60 SSIDExsSIKRKRRI (SEQ ID Swi4 113 kDa. KCII sites are NO:333) upstream of NLS like in SV4O large T; the homologous prokaryotic MutS and HexA lack NLS 96 GELAKRVARHOKARE 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 US 2003/0072794 A1 Apr. 17, 2003 36

TABLE 8-continued Nuclear localization signals on DNA repair proteins Gene Equivalent protein Putative NLS product in other species Features involved in recombination repair. None Rhp3 or ERCC2 772 aa; DNA helicase: 65% 681 DKRYGRSDKRTKLPK radi15 RAD3 identity to RAD3 and 55% (SEQ ID NO:337) identity to ERCC2; essential for viability 464 PPSKRRRVRGG Radlö RAD 1 Function in repair of UV (SEQ ID NO:338) damage for both cyclobutane dimer and (6-4) photoproduct lesions; Rad 16 interacts with SwiO. 431 DFKOAILRKRKNESPE Rad21 628 aa, 67.8 kDa, acidic EVEP (SEQ ID NO:339) protein; a single base substitution in mutant rad21 45, changing an Ile into a Thr, is responsible for the low efficiency in repair of DSBs after g-radiation although capable of arresting atG2. 490 DKKAKKG (SEQ ID Rad22 RAD52 496 aa; functions in NO:340) recombination-repair and mating-type switching. 394 DVVOFYLKKKYTRSKRN Rad32 MRE11 (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 Rhp51 (RAD51). Rad51 recombination-repair GLAKKYRDHKTHLHIP (weak Hhp1 CKI (mamm) 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; GLAKKYRD KTHVHIP (H in HRR25 (S. cer) mutation in this gene causes Hhp1 is replaced by F in Hhp2) repair defects (SEQ ID NO:344)

0186 TABLE 9-continued TABLE 9 NLS in Transcription factors NLS in Transcription factors NLS and Flanks Protein factor and features NLS and Flanks Protein factor and features (SEQ ID NO:355) erythroleukemia cells). Related highly basic OKGNRKKM to ets oncogene HRQRTRKR Human GCF (GC-factor) (SEQ ID NO:356) (SEQ ID NO:345) WKKVKKKL LRRKSRP (SEQ ID NO:357) (SEQ ID NO:346) WKRKK Human PRDII-BF1 that binds SRRTKRRO (SEQ ID NO:358) to IFN-B gene promoter. (SEQ ID NO:347) CRNRYRKLE (The largest DNA-binding GRKRKKRT Oct.-6 protein transcription (SEQ ID NO:359) protein known, of 298 kD). (SEQ ID NO:348) factor from mouse cells IRKRRKMK GRRRKKRT Mouse Oct-2 protein transcription (SEQ ID NO:360) (SEQ ID NO:349) factors (Oct-2.1 for Oct-2.6 isoforms) PKKKRLRL ARKRKRT Oct-3 from mouse P19 (SEQ ID NO:361) (SEQ ID NO:350) embryonal carcinoma cells GKKKKRKREKL Murine LEF-1 (397 aa). NRROKGKRS (within the Lymphoid-specific with an (SEQ ID NO:351) HMG-box) HMG1-like box. NLS is identical ECRRKKKE Human ATF-1. In basic (SEQ ID NO:362) to that of human TCF-1C. (SEQ ID NO:352) regionfleucine zipper. GKKKKRKREKL Human TCF-1C (399 aa) ERKKRRRE Human ATF-3 (in basic region (within the HMG-box) (T cell-specific transcription factor that (SEQ ID NO:353) that binds DNA) HMG-box) activates the T cell receptor AKCRNKKKEKT (SEQ ID NO:363) CC). Contains an HMG box. (SEQ ID NO:354) NLS core is identical to SKKKRL Mouse Pu. 1 (Friend that of murine LEF-1. US 2003/0072794 A1 Apr. 17, 2003 37

TABLE 9-continued TABLE 9-continued NLS in Transcription factors NLS in Transcription factors NLS and Flanks Protein factor and features NLS and Flanks Protein factor and features GKKKRRSREKH Human TCF-1 Group 0000 (within the (uniquely T cell-specific). (SEQ ID NO:364) HMG box containing. DRNKKKKE Xenopus RAR (retinoic acid receptor) PKKCRARF (SEQ ID NO:394) (SEQ ID NO:365) ARRRRP FKORRIKL Xenopus laevis Oct-1 (within (SEQ ID NO:395) (SEQ ID NO:366) POU-domain) GRRRRA Human ATF-2 (the 2nd and NRRRKKRT (SEQ ID NO:396) 3rd NLS are in basic region (SEQ ID NO:367) DEKRRKV that binds DNA) NRROKEKRI (SEQ ID NO:397) (SEQ ID NO:368) CROKRKV DKRSRKRKRSK Drosophila Suvar (3) 7 gene product (SEQ ID NO:398) (SEQ ID NO:369) involved in position-effect ERKRRD Myn (murine homolog of Max). Forms RLRIDRKRN variegation (932 aas). Five widely (SEQ ID NO:399) a specific DNA-binding (SEQ ID NO:370) spaced zinc-fingers could help SRKKLRME complex with c-Myc oncoprotein AKRSRRS condensation of the chromatin fiber. (SEQ ID NO:400) through a helix-loop-helix/ (SEQ ID NO:371) leucine zipper. IRKRRKMKSVGDE, Human MBP-1 (class I MHC enhancer EEKRKRTYE human NFkB p65 (550 aa). (SEQ ID NO:372) binding protein 1) mw 200 kD. (SEQ ID NO:401) Not binding DNA; complexed (not suggested Induced by phorbol esters and with p50 that binds DNA. NFkB p50 as NLS by the mitogens in Jurkat T cells. also contains a NLS (Table 3b). authors; between GRRRRA Human HB16, a cAMP response the 1st and 2nd (SEQ ID NO:402) element-binding protein Zinc finger) DEKRRKF PPKKKRLRLAE (SEQ ID NO:403) (suggested as NLS by the SRCROKRKV authors; just before 2nd zinc (SEQ ID NO:404) finger) SKKKKTKV Human TFIIE-? (general (SEQ ID NO:373) (SEQ ID NO:405) transcription initiation CRNRYRKLE NRPDKKK protein factor; forms (within 1st zinc finger) (SEQ ID NO:406) tetramer C.B., with TFIIE-C) (SEQ ID NO:374) ORRKKP PRRKRRV rat TTF-1 (thyroid nuclear factor (SEQ ID NO:407) (SEQ ID NO:375) hat binds to the promoter of OKKRRFKT HRYKMKRO hyroid-specific genes). An (SEQ ID NO:408) (SEQ ID NO:376) homeodomain protein. SRKRKM Human kup transcriptional activator DGKRKRKN Human thyroid hormone receptor C. (SEQ ID NO:409) (433 aas). Two distantly spaced (SEQ ID NO:377) (c-erbA-1 gene). Belongs to the Zinc fingers. Expressed in DDSKRVAKRKL amily of cytoplasmic proteins that hematopoietic cells and testis. (SEQ ID NO:378) are receptors of hydrophobic ERKRLRNRLA Mouse Jun-B homologue to NRERRRKEE igands such as steroids, vitD, (SEQ ID NO:410) avian sarcoma virus 17 oncogene (SEQ ID NO:379) retinoic acid, thyroid hormones. v-jun product. One region is similar WKORRKF The ligand binding may expose the NLS ATKCRKRKL to yeast GCN4 and to Fos. or nuclear import of the (SEQ ID NO:411) (SEQ ID NO:380) receptor-ligand complex. (19 aa stretch) NRRKRKRS Drosophila gcl (germ cell-less) gene DKRXERKRRD (N-terminus) Max (specifically associates with (SEQ ID NO:381) product (569 aa, 65 kD), located (SEQ ID NO:412) c-Myc, N-Myc, L-Myc). The Max-Myc PKKKKL in nuclei, required for germ OSRKKLRME complex binds to DNA; neither (SEQ ID NO:382) ine formation. (C-terminus) Max nor Myc alone exhibit ARRKRRRL C. elegans Sdc-3 protein (SEQ ID NO:413) appreciable DNA binding. (SEQ ID NO:383) (sex-determining protein) DKEKKKLEEDE Chicken VBP (vitellogenin gene LKFKKVRD (2,150 aas). A zinc finger protein. (within an binding protein). Leucine zipper. (SEQ ID NO:384) acidic region) Related to rat DBP. FKKFRKF (SEQ ID NO:414) (SEQ ID NO:385) IKKAKKV GKOKRRF (SEQ ID NO:415) (SEQ ID NO:386) TRRKKN ERLKRDKEKREKE (SEQ ID NO:416) (SEQ ID NO:387) TRDDKRRA Xenopus borealis B1 factor. Closely TRGRPKKVKE (SEQ ID NO:417) related to the mammalian USF. (SEQ ID NO:388) EVERRRRDK Binds to CACGTG in TFIIIA promoter SKKRGRRRKKT (SEQ ID NO:418) to developmentally regulate its expression. (SEQ ID NO:389) TRDEKRRA Human USF (upstream stimulatory TRROKRAKV (SEQ ID NO:419) factor) activating the major late (SEQ ID NO:390) EVERRRRDK adenovirus promoter SRKSKKRLRA (SEQ ID NO:420) (SEQ ID NO:391) YRRYPRRRG YB-1, a protein that binds to the MHC LKKRRKIKNK Drosophila BBF-2 (related to (SEQ ID NO:421) class II Ybox. YB-1 is a negative (SEQ ID NO:392) CREB/ATF) ORRPYRRRRF regulator. ESRRKKKE (SEQ ID NO:422) (SEQ ID NO:393) YRPRFRRG US 2003/0072794 A1 Apr. 17, 2003 38

TABLE 9-continued TABLE 9-continued NLS in Transcription factors NLS in Transcription factors NLS and Flanks Protein factor and features NLS and Flanks Protein factor and features (SEQ ID NO:423) Group 000x00 ORRYRRN (SEQ ID NO:424) DKRRKN Human glucocorticoid receptor YRRRRP (SEQ ID NO:451) (SEQ ID NO:425) ARKTKKK AKEROKKD Human TFEB Binds to IgH enhancer. (SEQ ID NO:452) (SEQ ID NO:426) 473 DKIRRKNCP Mouse and human GR ERRRRF (SEQ ID NO:453) (glucocorticoid recptor) (SEQ ID NO:427) EARKTKKKIKGIO LKEROKKD Human TFE3 (536 aa). Binds to uE3 (SEQ ID NO:454) (SEQ ID NO:428) enhancer of IgH genes. Group 000x0 IERRRRFN (SEQ ID NO:429) YRVRRERN C/EBP (CCAAT/enhancer YFRRRRLEKD (SEQ ID NO:455) binding protein). (SEQ ID NO:430) VRKSRDKA Functions in liver-specific KTVALKRRLKASSRL Human Dr1 (176 aa, 19 kD). (SEQ ID NO:456) gene expression. (SEQ ID NO:431) Interacts with TBP (TATA-binding DRLRKRVE protein) thus inhibiting association (SEQ ID NO:457) of TFILA and/or TFIIB with TBP. DKRRKN Human mineralocorticoid receptor TBP-Dr1 association is affected by (SEQ ID NO:458) Dr1 phosphorylation to repress ARKSKKL activated and basal transcription. (SEQ ID NO:459) 1 LRRRGROTY Drosophila ultrabithorax DKRRKN Human PR (progesterone receptor) (SEQ ID NO:432) protein (from the conserved 61 (SEQ ID NO:460) 27 LTRRRRIEM amino acid homeodomain segment GRKFKKF (SEQ ID NO:433) only). Conserved in the antenappedia (SEQ ID NO:461) Syst homeodomam protein. EEVORKROKLMP Human and mouse NFkB 105 kD SNRRRPDHR C. elegans sex-determining Tra-1 (SEQ ID NO:462) gets (968 aas) (first R is at SESSEO ID NO:435 Enrotein. larvalZinc finger.I Peaks in the EEVORKROKL Human NF-kB p50 (DNA-binding (SEQ ID NO:436) (SEQ ID NO:463) subunit). Identical to protein PAPRRRRSADNKD, KBF1, homologous to rel oncogene (SEQ ID NO:437) product. NF-kB p65 also PKKPRHOF contains a NLS (Table 3a). (SEQ ID NO:438) GKTRTRKO Human TEF-1 (SV40 transcriptional EKRKKERN Yeast NPS1 transcription protein (SEQ ID NO:464) enhancer factor 1). 426 aa. (SEQ ID NO:439) factor (1359 aa) involved in cell ARRKSRD LLRRLKKEVE growth control at G2 phase. (SEQ ID NO:465) (SEQ ID NO:440) Has a catalytic domain of ORKERKSKS Rat, mouse, human IRF-1 (interferon protein kinases. (SEQ ID NO:466) regulatory factor-1). Induced in EPLGRIRQKKRVY.D., TKSKTKRKL lymphoma T cells by the pituitary (SEQ ID NO:441) (SEQ ID NO:467) peptide hormone prolactin. (EDAIKKRREARERRRLRQ) Regulates the growth-inhibitory (SEQ ID NO:442) interferon genes. DKETTASRSKRRSSRKKRT GKCRKKN Ehrlich ascites S-II transcription factor. (SEQ ID NO:443) (SEQ ID NO:468) A general factor that acts at ESKKKKPKL the elongation step. (SEQ ID NO:444) ERSKKRSRE Tobacco TAF-1 transcriptional activator KKTAAKKTKTKS (SEQ ID NO:469) (SEQ ID NO:445) ERELKREKRKO ORKROKL Human 243 transcriptional (SEQ ID NO:470) (SEQ ID NO:446) activator (968 aas), induced ARRSRLRKO KAKKOK by mitogens (SEQ ID NO:471) (SEQ ID NO:447) in T cells. N-terminal half is YKLDHMRRRIETDE Drosophila TFIIEC (433 aa), a general LRRKROK homologous to oncoprotein Rel and (SEQ ID NO:472) transcription factor for RNA (SEQ ID NO:448) Drosophila Dorsal protein polymerase II. Composed of involved in development. The C- subunits C. and 3. terminal half contains repeats DKNRRKS Human ER (estrogen receptor); 595 aa. found in proteins involved in (SEQ ID NO:473) cell-cycle control of yeast and IRKDRRG tissue differentiation in Drosophila. (SEQ ID NO:474) RDIRRRGKNKV Mouse NF-E2 (45 kD), an erythroid KRSKKN (SEQ ID NO:449) transcription factor from mouse (SEQ ID NO:475) erythroleukemia (MEL) cells. Involved EORREHRIE Yeast ADA2 (434 aa), a potential in globin gene regulation. (SEQ ID NO:476) transcriptional adaptor required ONCRKRKLE Binds to AP-1-1ike sites. TTRAEKKRLL for the function of certain (SEQ ID NO:450) Homology to Jun B, GCN4, Fos, (SEQ ID NO:477) acidic activation domains. ATF1 and CREB in basic region/leucine DKKRSKEAKE zipper (see FIG. 2). (SEQ ID NO:478) US 2003/0072794 A1 Apr. 17, 2003 39

TABLE 9-continued TABLE 9-continued NLS in Transcription factors NLS in Transcription factors

NLS and Flanks Protein factor and features NLS and Flanks Protein factor and features EAALRRKIRTISK Yeast GCN5 gene product (439 aa), YRORRER Ig/EBP-1 (immunoglobulin gene (SEQ ID NO:479) required for the function of GCN4 transcriptional activator and for (SEQ ID NO:508) enhancer-binding protein). Forms the activity of the HAP2-3-4 complex. VKKSRLKSKOK heterodimers with CEBP. Group 00x00 (SEQ ID NO:509) EDPEKEKRIKELE mouse c-Myb NKKMRRNRF Mouse LFB3 (SEQ ID NO:510) (SEQ ID NO:480) MRRKV NRRKx. ROK (SEQ ID NO:511) (SEQ ID NO:481) DYYKVKRPKTD Drosophila eyes absent protein (760 aa), TKKGRRNRF Mouse LFB1 (SEQ ID NO:512) a nuclear protein that functions in (SEQ ID NO:482) GRARGRREHO early development to prevent NRRKx RHK (SEQ ID NO:513) programmed cell death and to allow the (SEQ ID NO:483) FRYRKIKDIY event that generate the eye to proceed. NKKMRRNRFK rat wNF1-A (SEQ ID NO:514) Mutations cause programmed cell death (SEQ ID NO:484) of eye progenitor cells. NKKMRRNR murine HNF-1B Group 0x0x00 (SEQ ID NO:485) TKKGRRNRF mouse HNF-1 AKAKAKKA (SEQ ID NO:486) rat IL-6DBP interacting with NKKMRRNRF human wNF1 (SEQ ID NO:515) interleukin-6 responsive elements. (SEQ ID NO:487) Has a leucine Zipper domain. TKKGRRNRF rat liver HNF1 DKRORNRC mouse H-2RIIBP (MHC class I genes (SEQ ID NO:488) (SEQ ID NO:516) H-2 region II binding protein). LRROKRFK Fkirtirk) Member of the nuclear hormone (SEQ ID NO:489) receptor superfamily. QQHSHQ Fkirtirk) chicken RXR, related to RAR (retinoic (SEQ ID NO:490) DKRORNRC acid receptor), a nuclear LRROKRFK rat HNF-3y (SEQ ID NO:517) protein factor from the thyroid/steroid (SEQ ID NO:491) hormone receptor family LRROKRFK at HNF-3C WKSKAKKT human NF-IL6 (345 aa). Specifically (SEQ ID NO:492) (SEQ ID NO:518) binds to IL1-responsive LKEKERKA rat DBP a protein factor that YKIRRERN element in the IL-6 gene. (SEQ ID NO:493) binds to the D site of the albumin (SEQ ID NO:519) Leucine zipper. Homology to C/EBP. MKKARKV gene promoter VRKSRDKA (SEQ ID NO:494) PRRERRY rat AT-BP1. Highly acidic domain. Two (SEQ ID NO:520) (SEQ ID NO:495) zinc fingers. Binds to the OKKNRNKC mouse PPAR (peroxisome proliferator B-domain of C1-antitrypsin gene (SEQ ID NO:521) activated receptor) promoter and to the NF-kB site in the Group 000xx00 MHC gene enhancer. DRRVRKGKV A 19 kD Drosophila melanogaster EOIRKLVKKHG yeast RAP1 (SEQ ID NO:496) nonhistone associated with (SEQ ID NO:522) It binds regulatory sites at heterochromatin. FRRSMKRKA yeast mating type silencers. SKHGRRARRLDP murine EBF (early B-cell factor) human vitamin D receptor (427 aa) (SEQ ID NO:497) of 591 aa. Regulates the pre-B and (SEQ ID NO:523) B lymphocyte-specific mb-1 gene. Group OOxx00 Expressed in pre-B and B-cell lines but not in plasmocytomas, LKRHORRH mouse WT1 (the murine homolog T-cell and nonlymphoid cell lines. (SEQ ID NO:524) of human Wilms tumor GRRTRRE human Sp1 predisposition gene WT1) (SEQ ID NO:498) LKRHORRH human WT33 (Wilms' tumor DEOKRAEKKAKE yeast SNF2, a transcriptional regulator (SEQ ID NO:525) predisposition) (SEQ ID NO:499) of many genes. Group 000xx0 IRRHKVRP (SEQ ID NO:500) LLRRLKKDVE LKESKRKYDE yeast SWI3 99 kD, highly (SEQ ID NO:501) (SEQ ID NO:526) acidic protein. Group 0x00x0 Global transcription activator. AKAKAKKA mouse AGP/EBP (87% similarity to EVLKVOKRRIYD human RBAP-1 (retinoblastoma (SEQ ID NO:502) C/EBP), ubiquitously expressed (SEQ ID NO:527) associated protein 1) factor (412 aa). YKMRRERN A protein that binds to the pocket (SEQ ID NO:503) VRKSRDKA (functional domain) of the (SEQ ID NO:504) retinoblastoma (RB) protein involved AKAKAKKA rat LAP, a 32-kD liver-enriched in suppression of cell growth (SEQ ID NO:505) transcriptional activator, also present (tumor suppressor). The transcription YKMRRERN in lung, with 71% sequence similarity factor E2F, implicated in cell (SEQ ID NO:506) to C/EBP. Leucine zipper. growth, binds to the same pocket of RB. VRKSRDKA Accumulates to maximal levels (SEQ ID NO:507) around birth. US 2003/0072794 A1 Apr. 17, 2003 40

0187)

TABLE 10 NLS in other nuclear proteins Putative NLS Protein YKSKKKA (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:531) Yeast L16 ribosomal protein GKGKYRNRRW (SEQ ID NO:532) yeast L2 ribosomal protein (homologous to Xenopus L1). Encoded by intronless genes. GKGKMRNRRRIORRG (SEQ ID NO:533) Xenopus laevis L1 ribosomal protein (homologous NKKVKRRELKKN (SEQ ID NO:534) to yeast L2) Encoded by intronless genes. AKTARRKA (SEQ ID NO:535) IKAKEKKP (SEQ ID NO:536) GKPKAKKP (SEQ ID NO:537) AKAKKRQ (SEQ ID NO:538) ERKRKS (SEQ ID NO:539) human S6 ribosomal protein (homologous to yeast GKRPRTKA (SEQ ID NO:540) S10) HKRRRI (SEQ ID NO:541) LKKQRTKKNKE (SEQ ID NO:542) PKMRRRTYR (SEQ ID NO:543) Rat L17 ribosomal protein (184 aas) KKKISQKKLKK (SEQ ID NO:544) YMRRRTYRA (SEQ ID NO:545) Podocoryne carnea (hydrozoan, Coelenteratum) EVKKVSKKKL (SEQ ID NO:546) L17 ribosomal protein (184 aas) highly homologous to rat L17. ERNRKDKDAKFR (SEQ ID NO:547) human, rat ribosomal S13 protein ERKRKS (SEQ ID NO:548) yeast S10 ribosomal protein (homologous to human QRLQRKRH (SEQ ID NO:549) S6) IRKRRA (SEQ ID NO:550) GRRRKKHRSRSRSRERRSRSRDRGRGGRER 35 kD subunit of U2 small nuclear DRRRSRDRER (SEQ ID NO:551) ribonucleoprotein auxiliary factor (U2AF), an essential mammalian splicing factor. U2AF interacts with the 65 kD subunit (U2AF). Both proteins are concentrated in a small number of subnuclear organelles, the coiled bodies. EFEDPRD (SEQ ID NO:552) 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 EERMERKRREK (SEQ ID NO:555) HRDRDRDRERERRESRERDKERERRRSRSRD RRRRSRSRDKEERRRSRERSKDKDRDRKRRS SRSRERARRERERKEE (SEQ ID NO:556) RDRDRERRRSHRSERERRRDRDRDRDRDREH KRGER (SEQ ID NO:557) QKRNNKKSKKKRCAE (SEQ ID NO:558) yeast TRM1 enzyme for the N, N'- 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 (70-213 aa segment) known to cause nuclear localization off-galactosidase. NKRKRV (SEQ ID NO:560) Yeast nucleoporin NUP1 (1076 aa, 113 kD); an SLKINRSNRKRE (SEQ ID NO:561) integral component of the pore complex. Involved EPKRKRRLP (SEQ ID NO:562) in both binding and translocation steps of nuclear ARMRHSKR (C-terminus) (SEQ ID NO:563) import. KAEKExKVD,E, (SEQ ID NO:564) Chicken, Xenopus No 38 nucleolar (38 kD); Kx.Kx. Kx.R (SEQ ID NO:565) involved in intranuclear packaging of preribosomal particles. Shuttles between nucleus and cytoplasm. KTEREAEKALEEKx.R (SEQ ID NO:566) Chicken, hamster nucleolin (92 kD). Binds Kx. Kx.Kx.RX3EDTTEETLR (SEQ ID NO:567) preribosomal RNA. Shuttles between nucleus and RGRGRGRGFGRGRGFGRGFRGRG cytoplasm. DHKPQGKKIKFE (SEQ ID NO:568) (C-terminus) WYKHFKKTKD (SEQ ID NO:569) human SATB1 (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. QKKKQMKAD (SEQ ID NO:570) yeast CBF5p, a centromere-binding protein (KKEKKE)s (SEQ ID NO:571) (55kDa, 483aa). The KKEKKRKSED (SEQ ID NO:572) KKE repeat at its C-terminus

US 2003/0072794 A1 Apr. 17, 2003 42

REFERENCES 0208 Boulikas, T. (1997c) “Nuclear localization sig nal peptides for the import of plasmid DNA in gene 0188 U.S. Patent Documents therapy'Int. J. Oncol. 10:301-309. 0189 U.S. Pat. No. 4,394.448 July, 1983 Szoka, Jr. et 0209 Boulikas, T. (1998a) “Status of gene therapy in al. 1997: Molecular mechanisms, disease targets, and 0190 U.S. Pat. No. 4,598,051 July, 1986 Papahad clinical applications'Gene Ther. Mol. Biol. 1:1-172. jopoulos et al. 0210 Boulikas, T. (1998b) “Nucleocytoplasmic traf ficking: implications for the nuclear import of plasmid 0191 U.S. Pat. No. 5,013,556 May, 1991 Woodle et al. DNA during gene therapy'Gene Ther. Mol. Biol. 1:713 0192 Journal Articles 740. 0193 Allen, T. M. and Chonn, A. (1987) “Large unila 0211 Boulikas, T. and Martin, F. (1997) “Histones, mellar liposomes with low uptake into the reticuloen protamine, and polylysine but not poly(E:K) enhance dothelial system” FEBS Lett. 223:42-46. transfection efficiency'Int. J. Oncol. 10:317-322. 0194 Allen, T. M. et al. (1991) “Liposomes containing 0212 Capaccioli, S. et al. (1993) “Cationic lipids Synthetic lipid derivatives of polyethylene glycol show improve antisense oligonucleotide uptake and prevent prolonged circulation half-lives in vivo’ Biochim. Bio degradation in cultured cells and in human Serum'Bio phys. Acta 1066:29-36. chem. Biophys. Res. Comm. 197:818-825. 0.195 Anderson, W. F. (1992) “Human gene therapy 0213 Creuzenet, C. et al. (1997) “Interaction of alpha Science 256:808–813. S2- and beta-casein signal peptides with DMPC and DMPG liposomes' Peptides 18:463-472. 0196) Aoki, K. et al. (1995) “Liposome-mediated in Vivo gene transfer of antisense K-ras construct inhibits 0214) Culver, K.W. (1996) in: Gene Therapy: Aprimer pancreatic tumor dissemination in the murine perito for physicians, Second Edition. Mary Ann Liebert, Inc. neal cavity"Cancer Res. 55:3810-3816. Publications, NY, pp. 1-198. 0215. Curtain, C. et al. (1999) “The interactions of the 0197) Arcasoy, S. M. et al. (1997) “Polycations N-terminal fusogenic peptide of HIV-1 gp41 with neu increase the efficiency of adenovirus-mediated gene tral phospholipids'Eur: Biophys. J 28:427-436. transfer to epithelial and endothelial cells in vitro'Gene Ther. 4:32-38. 0216) de la Maza, A. et al. (1998) “Solubilization of phosphatidylcholine lipoSomes by the amphoteric Sur 0198 Beauchamp, C. O. et al. (1983) “A new proce factant dodecyl betaine''Chem. Phys. Lipids 94:71-79. dure for the Synthesis of polyethylene glycol-protein adducts, effects on function, receptor recognition, and 0217 Decout, A. et al. (1999) “Contribution of the clearance of Superoxide dismutase, lactoferrin, and hydrophobicity gradient to the Secondary Structure and alpha 2-macroglobulin’Anal. Biochem. 131:25-33. activity of fusogenic peptides' Mol. Membr. Biol. 16:237-246. 0199 Bongartz, J.-P. et al. (1994) “Improved biologi cal activity of antisense oligonucleotides conjugated to 0218. Duguid, J. G. et al. (1998) “A physicochemical approach for predicting the effectiveness of peptide a fusogenic peptide’Nucl. Acids Res. 22:4681-4688. based gene delivery Systems for use in plasmid-based 0200 Boulikas, T. (1993) “Nuclear localization signals gene therapy’ BiophyS. J. 74:2802-2814. (NLS)'Crit. Rev. Eukar. Gene Expression 3:193-227. 0219. Filion, M. C. and Phillips, N. C. (1997) “Tox 0201 Boulikas, T. (1994) “Putative nuclear localiza icity and immunomodulatory activity of liposomal vec tion signals (NLS) in protein transcription factors'J. tors formulated with cationic lipids toward immune Cell. Biochen. 55:32–58. effector cells'Biochim. Biophys. Acta 1329:345-356. 0220 Fresta, M. et al. (1998) “Liposomal delivery of 0202 Boulikas, T. (1996a) “Cancer gene therapy and a 30-mer antisense oligodeoxynucleotide to inhibit immunotherapy'Intl. J. Oncol. 9:941-954. proopiomelanocortin expression'J. Pharm. Sci. 0203 Boulikas, T. (1996b) “Gene therapy to human 87:616-625. diseases: ex vivo and in vivo studies' Intl. J. Oncol. 0221) Gabizon, A. and Papahadjopoulos, D. (1988) 9:1239-1251. "Liposome formulations with prolonged circulation 0204 Boulikas, T. (1996c) “Liposome DNA delivery time in blood and enhanced uptake by tumors'Proc. and uptake by cells'Oncol. Rep. 3:989-995. Natl. Acad. Sci. USA 85:6949-6953. 0205 Boulikas, T. (1996d) “Nuclear import of protein 0222 Gabizon, A. et al. (1989) “Pharmacokinetics and kinases and cyclins'J. Cell. Biochem. 60:61-82. tissue localization of doxorubicin encapsulated in stable liposomes with long circulation times J. Natl. 0206 Boulikas, T. (1997a) “Gene therapy of prostate Cancer InSt. 81:1484-1488. cancer: p53, Suicidal genes, and other targets'Antican cer ReS. 17:1471-1506. 0223) Ghosh, J. K. and Shai, Y. (1999) “Direct Evi dence that the N-Terminal Heptad Repeat of Sendai 0207 Boulikas, T. (1997b) “Nuclear import of DNA Virus Fusion Protein Participates in Membrane repair proteins'Anticancer Res. 17:843-864. Fusion.J. MOI. Biol. 292:531-546. US 2003/0072794 A1 Apr. 17, 2003

0224) Green, M. and Loewenstein, P. M. (1988) 0239 Lins, L. et al. (1999) “Molecular determinants of "Autonomous functional domains of chemically Syn the interaction between the C-terminal domain of thesized human immunodeficiency virus tat transacti Alzheimer's beta-amyloid peptide and apolipoprotein vator protein'Cell 55:1179-1188. E alpha-helices'J. Neurochem. 73:758-769. 0225 Gupta, D. and Kothekar, V. (1997) “500 pico 0240 Litzinger, D. C. et al. (1996) “Fate of cationic Second molecular dynamics simulation of amphiphilic liposomes and their complex with oligonucleotide in polypeptide Ac(LKKL), NHEt with 1.2 di-mySristoyl vivo"Biochim. Biophys. Acta 1281:139-149. sn-glycero-3-phosphorylcholine (DMPC) molecules 0241 Lopez, O. et al. (1998) “Direct formation of "Indian J. Biochem. Biophys. 34:501-511. mixed micelles in the solubilization of phospholipid 0226 Hofland, H. E. J. et al. (1996) “Formation of liposomes by Triton X-100” FEBS Lett. 426:314-318. Stable cationic lipid/DNA complexes for gene transfer 0242 Lusa, S. et al. (1998) “Direct observation of Proc. Natl. Acad. Sci. USA 93:7305-73.09. lipoprotein cholesterol ester degradation in lySOSomes 0227 Jaaskelainen, I. et al. (1994) “Oligonucleotide Biochen. J 332:451-457. cationic liposome interactions. A physicochemical 0243 Macosko, J. C. et al. (1997) “The membrane study"Biochim. Biophys. Acta 1195:115-123. topology of the fusion peptide region of influenza 0228 Judice, J. K. et al. (1997) “Inhibition of HIV type hemagglutinin determined by spin-labeling EPR'J. 1 infectivity by constrained alpha-helical peptides: MOI. Biol. 267: 1139-1148. implications for the viral fusion mechanism'Proc. Natl. 0244 Macreadie, I. G. et al. (1997) “Cytotoxicity Acad. Sci. USA 94:13426-13430. resulting from addition of HIV-1 Nef N-terminal pep 0229) Kono, K. et al. (1993) “Fusion activity of an tides to yeast and bacterial cells' Biochem. BiophyS. amphiphilic polypeptide having acidic amino acid resi ReS. Commun. 232:707-711. dues: generation of fusion activity by alpha-helix for 0245 Martin, F. and Boulikas, T. (1998) “The chal mation and charge neutralization'Biochim. BiophyS. lenge of liposomes in gene therapy'Gene Ther. Mol. Acta 1164:81-90. Biol 1: 173-214. 0230. Lambert, G. et al. (1998) “The C-terminal helix 0246 Martin, I. et al. (1999) “Membrane fusion of human apolipoprotein AII promotes the fusion of induced by a short fusogenic peptide is assessed by its unilamellar liposomes and displaces apolipoprotein AI insertion and orientation into target bilayers'Biochem from high-density lipoproteins'Eur: J. Biochem. istry 38:9337-9347. 253:328-338. 0247 Martin, I. and Ruysschaert, J. M. (1997) “Com 0231 Lambert, O. et al. (1998) “A new “gel-like” parison of lipid vesicle fusion induced by the putative phase in dodecyl maltoside-lipid mixtures: implications fusion peptide of fertilin (a protein active in Sperm-egg in solubilization and reconstitution studies’ BiophyS. J. fusion) and the NH2-terminal domain of the HIV2 74:918-930. gp41” FEBS Lett. 405:351-355. 0232) Lappalainen, K. et al. (1997) “Intracellular dis 0248 Massari, S. and Colonna, R. (1986) “Gramicidin tribution of oligonucleotides delivered by cationic lipo induced aggregation and size increase of phosphatidyl Somes: light and electron microscopic Study'His choline vesicles"Chem. Phys. Lipids 39:203-220. tochem. Cytochem. 45:265-274. 0249 Melino, S. et al. (1999) “Zn(2+) ions selectively 0233 Lasic, D. (1997) in: Liposomes in Gene Deliv induce antimicrobial Salivary peptide histatin-5 to fuse ery, CRC Press, pp. 1-295. negatively charged vesicles. Identification and charac 0234) Lee, S. et al. (1992) “Effect of amphipathic terization of a zinc-binding motif present in the func peptides with different alpha-helical contents on lipo tional domain"Biochemistry 38:9626-9633. some-fusion Biochim. Biophys. Acta 1103: 157-162. 0250 Midoux, P. and Monsigny, M. (1999) “Efficient 0235) Lelkes, P.I. and Lazarovici, P. (1988) “Pardaxin gene transfer by histidylated polylysine/pDNA com induces aggregation but not fusion of phosphati plexes’ Bioconjug. Chem. 10:406-411. dylserine vesicles” FEBS Lett. 230:131-136. 0251 Murata, M. et al. (1991) “Modification of the 0236 Leonard, A. N. and Cohen, D. E. (1998) “Sub N-terminus of membrane fusion-active peptides blockS micellar bile Salts Stimulate phosphatidylcholine trans the fusion activity’Biochem. BiophyS. Res. Commun. fer activity of sterol carrier protein 2' J. Lipid Res. 179:1050-1055. 39:1981-1988. 0252) Nidome, T. et al. (1997) “Membrane interaction of Synthetic peptides related to the putative fusogenic 0237 Lewis, J. G. et al. (1996) “A serum-resistant cytofectin for cellular delivery of antisense oligode region of PH-30 alpha, a protein in Sperm-egg fusion'J. oxynucleotides and plasmid DNA” Proc. Natl. Acad. Peptide Res. 49:563-569. Sci. USA 93:3176-3181. 0253 Pak, C. C. et al. (1999) “Elastase activated 0238 Li, S. and Huang, L. (1997) “In vivo gene liposomal delivery to nucleated cells'Biochim. Bio transfer via intravenous administration of cationic phys. Acta 1419:111-126. lipid-protamine-DNA (LPD) complexes"Gene Ther. 0254 Papahadjopoulos, D. et al. (1991) “Sterically 4:891-900. Stabilized liposomes: Improvements in pharmacokinet US 2003/0072794 A1 Apr. 17, 2003 44

ics and antitumor therapeutic efficacy'Proc. Natl. 0270 Song, Y. K. et al. (1997) “Characterization of Acad. Sci. USA 88:11460-11464. cationic liposome-mediated gene transfer in Vivo by 0255 Parente, R. A. et al. (1988) “pH-dependent intravenous administration'Hum. Gene Ther. 8:1585 fusion of phosphatidylcholine Small vesicles. Induction 1594. by a synthetic amphipathic peptide'J. Biol. Chem. 0271 Sorgi, F. L. et al. (1997) “Protamine sulfate 263:4724-4730. enhances lipid-mediated gene transfer'Gene Ther. 0256 Partidos, C. D. et al. (1996) “Priming of measles 4:961-968. Virus-specific CTL responses after immunization with a 0272 Suenaga, M. et al. (1989) “Basic amphipathic CTL epitope linked to a fusogenic peptide'Virology helical peptides induce destabilization and fusion of 215:107-110. acidic and neutral liposomes’ Biochim. Biophys. Acta 0257 Pecheur, E. I. et al. (1997) “Membrane anchor 981:143-150. age brings about fusogenic properties in a short Syn 0273 Takle, G. B. et al. (1997) “Delivery of oligori thetic peptide"Biochemistry 36:3773-3781. bonucleotides to human hepatoma cells using cationic 0258 Peelman, F. et al. (1999) “Characterization of lipid particles conjugated to ferric protoporphyrin IX functional residues in the interfacial recognition (heme)'Antisense Nucleic Acid Drug Dev. 7:177-185. domain of lecithin cholesterol acyltransferase (LCAT 0274) Templeton, N. S. et al. (1997) “Improved DNA: )"Protein Eng. 12:71-78. liposome complexes for increased Systemic delivery 0259 Pereira, F. B. et al. (1997) “Permeabilization and and gene expression'Nature Biotechnol. 15:647-652. fusion of uncharged lipid vesicles induced by the HIV-1 0275] Thierry, A.R. and Dritschilo, A. (1992) “Intrac fusion peptide adopting an extended conformation: ellular availability of unmodified, phosphorothioated dose and sequence effects’ BiophyS. J. 73:1977-1986. and liposomally encapsulated oligodeoxynucleotides 0260 Pillot, T. et al. (1999) “The nonfibrillar amyloid for antisense activity'Nucl. Acids Res. 20:5691-5698. beta-peptide induces apoptotic neuronal cell death: 0276 Tirosh, 0. et al. (1998) “Hydration of polyethyl involvement of its C-terminal fusogenic domain'J. ene glycol-grafted liposomes’ BiophyS. J. 74:1371 Neurochem. 73:1626-1634. 1379. 0261) Pillot, T. et al. (1997) “Specific modulation of 0277 Torchilin, V. P. (1998) “Polymer-coated long the fusogenic properties of the Alzheimer beta-amyloid circulating microparticulate pharmaceuticals'J. peptide by apolipoprotein E isoforms'Eur: J. Biochem. Microencapsul. 15:1-19. 243:650-659. 0278 Torchilin, V. P. et al. (1992) “Targeted accumu 0262 Pillot, T. et al. (1997) “The 118-135 peptide of lation of polyethylene glycol-coated immunoliposomes the human prion protein forms amyloid fibrils and in infarcted rabbit myocardium'FASEBJ 6:2716-2719. induces liposome fusion'J. Mol. Biol. 274:381-393. 0263 Plank, C. et al. (1996) “Activation of the 0279 Toumois, H. et al. (1990) “Gramicidin A induced complement System by Synthetic DNA complexes: a fusion of large unilamellar dioleoylphosphatidylcho potential barrier for intravenous gene delivery'Hum. line vesicles and its relation to the induction of type II Gene Ther. 7:1437-1446. nonbilayer structures'Biochemistry 29:8297-8307. 0264 Rodriguez-Crespo, I. et al. (1994) “Prediction of 0280 Ulrich, A. S. et al. (1999) “Ultrastructural char a putative fusion peptide in the S protein of hepatitis B acterization of peptide-induced membrane fusion and virus.J. Gen. Virol. 75:637-639. peptide self-assembly in the lipid bilayer'BiophyS. J. 77:829-841. 0265 Rodriguez-Crespo, I. et al. (1999) “Fusogenic activity of hepadenavirus peptides corresponding to 0281 Voneche, V. et al. (1992) “The 19-27 amino acid Sequences downstream of the putative cleavage site'Wi Segment of gp51 adopts an amphiphilic structure and rology 261:133-142. plays a key role in the fusion events induced by bovine leukemia virus.J. Biol. Chen. 267:15,193-15197. 0266 Ross, G. et al. (1996) “Gene therapy in the United States: a five-year status report'Hum. Gene 0282) Wattiaux, R. et al. (1997) “Cationic lipids desta The 7:1781-1790. bilize lysosomal membrane in vitro'FEBS Lett. 417:199-2O2. 0267 Schroeder, F. et al. (1990) “Intermembrane cho lesterol transfer: role of Sterol carrier proteins and 0283 Weissig, V. et al. (1998) “Accumulation of pro phosphatidylserine'Lipids 25:669-674. tein-loaded long-circulating micelles and liposomes in Subcutaneous Lewis lung carcinoma in mice'Pharm. 0268 Schroth-Diez, B. et al. (1998) “Fusion activity of ReS. 15:1552-1556. transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin' J. 0284 Zelphati, O. and Szoka, Jr., F. C. (1997) “Intra Virol. 72:133-141. cellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids'Pharm. 0269 Schutze, W. and Muller-Goymann, C. C. (1998) "Phase transformation of a liposomal dispersion into a ReS. 13:1367-1372. micellar solution induced by drug-loading'Pharm. Res. 0285) Zuidam, N.J. and Barenholz, Y. (1997) “Elec 15:538-543. troStatic parameters of cationic liposomes commonly US 2003/0072794 A1 Apr. 17, 2003 45

used for gene delivery as determined by 4-heptadecyl 14. The method of claim 13, wherein the NLS peptide is 7-hydroxycoumarin’ Biochim. Biophys. Acta 1329:211 a peptide Selected from the group consisting of Seq. ID NOS. 222. 20-622. 15. The method of claim 1, wherein the fusogenic What is claimed is: karyophilic peptide conjugate is a Sole fusogenic peptide. 1. A method for producing micelles with entrapped thera 16. The method of claim 1, wherein the NLS peptide peutic agents, comprising: component of the fusogenic-karyophilic peptide conjugate is an NLS peptide Selected from the group consisting of Seq. a) combining an effective amount of a negatively charged ID NOS. 20-622. therapeutic agent with an effective amount of a cationic 17. The method of claim 1, wherein the fusogenic/NLS lipid in a ratio where about 30% to about 90% the peptide conjugates comprise amino acid Sequences Selected negatively charged atoms are neutralized by positive from the group consisting of (KAWLKAF) (SEQID NO:1), charges on lipid molecules to form an electroStatic GLFKAAAKLLKSLWKLLLKA (SEQ ID NO:2), micelle complex in about 20% to about 80% ethanol; LLLKAFAKLLKSLWKLLLKA (SEQ ID NO:3) as well as and all derivatives of the prototype b) combining the micelle complex of step a) with an (Hydrophobic Karyophilic, Hydrophobic-Karyophilicles effective amount of a fusogenic-karyophilic peptide where Hydrophobic is any of the A., I, L, V, P, G, W, F and conjugates in a ratio range of about 0.0 to about 0.3, Karyophilic is any of the K, R, or H, containing a positively thereby producing micelles with entrapped therapeutic charged residue every 3rd or 4th amino acid, that form alpha agents. helices and direct a net positive charge to the same direction 2. The method of claim 1, wherein the negatively charged of the helix. therapeutic agent is a therapeutic agent Selected from the 18. The method of claim 1, wherein the fusogenic/NLS group consisting of a polynucleotide and a negatively peptide conjugate comprise an amino acid Sequence Selected charged drug. from the group consisting of GLFKAIAGFIKNGWKG MIDGGGYC (SEQ ID NO:4) from influenza virus hemag 3. The method of claim 2, wherein the polynucleotide is glutinin HA-2 and YGRKKRRQRRR (SEQID NO:5) from a DNA polynucleotide or an RNA polynucleotide. TAT of HIV. 4. The method of claim 2, wherein the polynucleotide is 19. The method of claim 1, wherein the fusogenic/NLS a DNA polynucleotide. peptide conjugate comprise an amino acid Sequence Selected 5. The method of claim 4, wherein the DNA polynucle from the group consisting of MSGTFGGILAGLIGLL(K/R/ otide comprises plasmid DNA. H) (SEQID NO:6), derived from the N-terminal region of 6. The method of claim 1, further comprising combining the S protein of duck hepatitis B virus but with the addition an effective amount of an anionic lipid in Step a). of one to Six positively-charged lysine, arginine or histidine 7. The method of claim 6, wherein the anionic lipid is residues, and combinations of these, GAAIGLAWIPYFG dipalmitoyl phosphatidylglycerol (DDPG) or a derivative PAA (SEQ ID NO:7) derived from the fusogenic peptide of thereof. the Ebola virus transmembrane protein; residues 53-70 8. The method of claim 4, further comprising combining (C-terminal helix) of apolipoprotein (apo) All peptide, the an effective amount of a DNA condensing agent Selected 23-residue fusogenic N-terminal peptide of HIV-1 trans from the group consisting of Spermine, Spermidine, polyl membrane glycoprotein gp41, the 29-42-residue fragment ySine, polyarginine, polyhistidine, polyornithine and mag from Alzheimer's beta-amyloid peptide, the fusion peptide nesium or a divalent metal ion. and N-terminal heptad repeat of Sendai virus, the 56-68 9. The method of claim 5, wherein the plasmid DNA helical Segment of lecithin cholesterol acyltransferase. comprises a Sequence encoding p53, HSV-tk, p21, Bax, Bad, 20. The method of any of claims 13 to 19, wherein the IL-2, IL-12, GM-CSF, angiostatin, endostatin and oncosta NLS peptide component in fusogenic/NLS peptide conju tin. gates are Synthetic peptides containing the above Said NLS 10. The method of claim 1, wherein the cationic lipids are but further modified by additional K, R, H residues at the selected from the group consisting of 3 B-(N-(N',N'-dim central part of the peptide or with P or G at the N- or ethylaminoethane)carbamoyl)cholesterol, dimethyldiocta C-terminus. decyl ammonium bromide (DDAB), N-1-(2,3-dimyristy 21. The method of claim 13, wherein the fusogenic loxy)propyl-N,N-dimethyl-N-(2-hydroxyethyl) ammonium peptide/NLS peptide conjugates are linked to each other bromide (DMRIE), 1,2-dimyristoyl-3-trimethylammonium with a short amino acid stretch representing an endogenous propane (DMTAP), dioctadecylamidoglycylspermine protease cleavage site. (DOGS), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethy 22. The method of claim 1, wherein the structure of the lammonium chloride (DOTMA), 1,2- dipalmitoyl-3-trim preferred prototype fusogenic/NLS peptide conjugate used ethylammonium propane (DPTAP), 1,2-disteroyl-3-trim in this invention is: PKKRRGPSP(L/A/I) (SEQ ID ethylammonium propane (DSTAP). NO:8) where (L/A/I) is a stretch of 12-20 hydrophobic 11. The method of claim 10, wherein the cationic lipids amino acids containing A, L., I, Y, W, F and other hydro are combined with the fusogenic lipid DOPE in a molar ratio phobic amino acids. from about 1:1 to about 2:1. 23. The method of claim 1, wherein the fusogenic/NLS 12. The method of claim 11, wherein the cationic lipids peptide conjugates are added to the mixture of DNA/cationic are combined with the fusogenic lipid DOPE in a molar ratio lipid and are incorporated into micelles. of 1:1. 24. The method of claim 1, further comprising combining 13. The method of claim 1, wherein the fusogenic an effective amount of an encapsulating lipid Solution to Step karyophilic peptide is an NLS peptide. b). US 2003/0072794 A1 Apr. 17, 2003 46

25. The method of claim 24, wherein the encapsulating 38. The method of claim 34 or 37, wherein the adminis lipid is a lipid comprising cholesterol (40%), dioleoylphos tration is intravenous administration or by injection. phatidylethanolamine (DOPE) (20%), palmitoyloleoylphos 39. A micelle with an entrapped DNA polynucleotide phatidylcholine (POPC) (12%), hydrogenated soy phos produced by the method of claim 9. phatidylcholine (HSPC) (10%), 40. A method for reducing tumor size in a Subject com distearoylphosphatidylethanolamine (DSPE) (10%), sphin prising administration of an effective amount of the micelle gomyelin (SM) (5%), and derivatized vesicle-forming lipid of claim 39 to the subject. M-PEG-DSPE (3%). 26. The method of claim 24, wherein the encapsulating 41. The method of claim 40, further comprising admin lipid is a liposome. istration of an effective amount of a Second therapeutic 27. The method of claim 26, wherein the liposomes agent, wherein the agent is Selected from the group consist comprises vesicle-forming lipids and between about 1 to ing of ganciclovir, 5-fluorocytosine, an antisense oligonucle about 7 mole percent of distearoylphosphatidyl ethanola otides a ribozyme, and a triplex-forming oligonucleotide mine (DSPE) derivatized with an effective amount of poly directed against genes that control the cell cycle or Signaling ethyleneglycol. pathways. 28. The method of claim 27, wherein the liposomes have 42. The method of claim 41, further comprising admin a selected average size of about 80 to about 160 nm. istration of an effective amount of a Second therapeutic 29. The method of claim 27, wherein the polyethyleneg agent, wherein the Second therapeutic agent is Selected from lycol has a molecular weight from about 1,000 to about the group consisting of adriamycin, angiostatin, azathio 5,000 daltons. prine, bleomycin, buSulfane, camptothecin, carboplatin, car 30. A micelle with an entrapped therapeutic agent pro mustine, chlorambucile, chlormethamine, chloroquinoxa duced by the method of claim 1. line Sulfonamide, cisplatin, cyclophosphamide, 31. A liposome encapsulated therapeutic agent produced cycloplatam, cytarabine, dacarbazine, dactinomycin, dauno by the method of claim 24. rubicin, didox, doxorubicin, endostatin, enloplatin, estra 32. The method of claim 31, wherein the therapeutic agent mustine, etoposide, extramustinephosphat, flucytosine, fluo further comprises regulation by a liver, Spleen or bone rodeoxyuridine, fluorouracil, gallium nitrate, hydroxyurea, marrow regulatory DNA sequence. idoxuridine, interferons, interleukins, leuprolide, lobaplatin, 33. The method of claim 32, wherein the regulatory DNA lomustine, mannomustine, mechlorethamine, mechlore Sequence is nuclear matrix DNA isolated from liver, Spleen thaminoxide, melphalan, mercaptopurine, methotrexate, or bone marrow cells. mithramycin, mitobronitole, mitomycin, mycophenolic 34. A method for delivering a therapeutic agent in Vivo, acid, nocodaZOle, oncoStatin, Oxaliplatin, paclitaxel, penta comprising administration of an effective amount of the mustine, platinum-triamine complex, plicamycin, predniso micelle of claim 30 to a subject. lone, prednisone, procarbazine, protein kinase C inhibitors, 35. The method of claim 34, wherein the therapeutic agent puromycine, Semustine, Signal transduction inhibitors, further comprises regulation by a tumor-specific regulatory Spiroplatin, Streptozotocine, Stromelysin inhibitors, taxol, DNA sequence. tegafur, telomerase inhibitors, teniposide, thalidomide, thia 36. The method of claim 35, wherein the tumor-specific miprine, thioguanine, thiotepa, tiamiprine, tretamine, triazi regulatory Sequence is nuclear matrix DNA isolated from quone, trifosfamide, tyrosine kinase inhibitors, uramustine, Specific tumor cells. Vidarabine, vinblastine, Vinca alcaloids, Vincristine, Vin 37. A method for delivering a therapeutic agent in Vivo, desine, Vorozole, Zeniplatin, Zeniplatin, and Zinostatin. comprising administration of an effective amount of the lipoSome encapsulated agent of claim 31 to the Subject.