MXPA00002076A - Receptor-mediated gene delivery using bacteriophage vectors - Google Patents

Abstract

Filamentous phage particles displaying a ligand on their surface are used to deliver a therapeutic gene to a cell. The ligand is a fusion protein with a phage capsid protein, covalently conjugated to phage particles, or complexed with modified phage particles.

Description

GENE SUPPLY MEASURED BY RECEIVERS USING BACTERIÓFAGOS VECTORS FIELD OF THE INVENTION The present invention generally relates to the delivery of genes, and more specifically, to the preparation and use of bacteriophages modified with ligands to deliver genes that alter the phenotype, function, expression of genes, or viability of a cell in a therapeutic form. BACKGROUND OF THE INVENTION In one approach, gene therapy attempts to target cells in a specific manner. Therefore, a therapeutic gene is bound in some way to a targeting molecule in order to deliver the gene to a target cell or tissue. Current methods usually involve the binding of a targeting molecule such as a ligand or antibody that recognizes an internment receptor in the pure DNA or in a virus of mammalian cells (e.g., adenovirus) that contain the gene wanted. When pure DNA is used, it can be condensed in vitro in a compact geometry to enter the cells. A polycation such as polylysine, is commonly used to neutralize the charge on DNA and condense into toroid structures. However, this condensation process is poorly understood and difficult to control, therefore, making the manufacture to form homogeneous gene therapy drugs is extremely complicated.

Mammalian viruses, in contrast, can pack DNA into uniform particles, but due to their complexity, they are difficult to manipulate genetically and the manufacture of viral particles to supply genes is costly and time consuming. Bacteriophages offer an attractive alternative as a natural method for condensing and packaging therapeutic DNA and, because of their simplicity, they are relatively easy to genetically manipulate (and retain their functions). In addition, because bacteriophages are extremely simple entities, the large-scale production of gene delivery vectors based on genes could be easier and less expensive than, for example, the production of viral vectors of mammals. Bacteriophages, such as lambda and filamentous phages, have occasionally been used in efforts to transfer DNA into mammalian cells. In general, it was found that lambda transduction is a relatively rare event and reporter gene expression was weak. In an effort to improve transduction efficiency, methods using calcium phosphate or liposomes (which do not specifically target the cell surface receptor) when used together with lambda. Gene transfer has been observed via lambda phages using calcium phosphate co-precipitation (Ishiura, M. et al., Mol.Cell. Biol., 2: 607-616, 1982), or via filamentous phages using DEAE- dextran or lipopolyamine (Yokoyama-Kobayashi and Kato, Biochem Biophys, Res. Comm. 192: 935-939, 1993; Yokoyama-Kobayashi and Kato, Anal. Biochem. 223. ^^ * »^^^^^^ *** g < & ^ 130-134, 1994). However, these methods of introducing DNA into mammalian cells are not practical for gene therapy applications, since the transfection efficiency tends to be low, non-specific and the transfection is not only annoying, but promiscuous with respect to to the type of cells. Therefore, more reliable means are required to direct the vectors to specific cells (or receptors) and to ensure a therapeutically useful degree for delivering the gene and expression, if the bacteriophages are formed into vectors useful in therapeutic applications. Attempts to target filamentous phages or cells using a fusion of a cyclic RGD peptide and a phage coat protein or a peptide shell protein fusion accomplish limited success. Although phages target and enter, phage gene expression is not expected or reported (Hart et al., J. Biol. Chem. 269: 12468-12474, 1994; Barry et al., Nature Med. 2: 299-305, 1996). Although it should generally be understood that the sequence of the RGD peptide used by Hart and others, binds to integrins, Hart describes the RGD-mediated uptake of phage as a process similar to the phagocytic absorption of the bacterium via the protein invasin (a protein of RGD); Adenoviruses use RGD-integrin together with ligand receptor binding for internment. Therefore, it is not clear that the RGD-integrin binding facilitates the entry of the peptide or protein fusion via an endosomal mechanism mediated by the receptor, a mechanism that has been shown to give superior results. Therefore, for gene delivery applications, the methods and therapeutic agents that are simple to carry out and manufacture, efficient and target specific cells would be very beneficial. Similarly, vectors that deliver therapeutically useful amounts of genes of interest via numerous routes of administration, including oral means, may be convenient. In response to these long-established needs, the present invention provides compositions and methods for delivering the gene using bacteriophages that express the ligand and carry a gene of interest, as well as providing other related advantages. SUMMARY OF THE INVENTION In one aspect, the invention provides a method for delivering genes, comprising: administering to a patient filamentous phage particles having a ligand on their surfaces, wherein the vector within the phage encodes the gene product under control of a promoter. In related aspects, the invention provides methods for treating tumors, smooth muscle cell diseases, or angiogenic diseases, which comprise administering a pharmaceutical composition to a patient, comprising a physiologically acceptable pH regulatory solution and filamentous phage particles having a ligand on its surfaces, where the phage genome encodes a product of therapeutic genes under the control of a promoter. In a preferred embodiment, the ligand is a polypeptide reagent with the FGF receptor, and in a more preferred embodiment, the ligand is FGF-2. In other embodiments, the ligand is an antibody and preferably a single chain antibody. The ligand can generally be fused with a phage capsid protein or chemically conjugated to form a covalent bond or by a sandwich method. Generally, the capsid protein for gene fusion, is the lll gene or the gene VIII. In a preferred embodiment, an endosomal escape portion is incorporated into the ligand or is exhibited by other means on the surface of the phage. The ligand or phage may also further comprise a nuclear localization sequence. In another preferred embodiment, the phage genome is a phagemid. In a preferred embodiment, the therapeutic gene product is selected from the group consisting of protein, ribozyme, and antisense oligonucleotide, and in other embodiments, the therapeutic gene product is a cytotoxic agent (e.g., ribosome which inactivates the protein, such as saporin) or is an antibody that binds HER2 / neu.

In other aspects, the invention provides a pharmaceutical composition comprising a physiologically acceptable pH regulatory solution and filamentous phage particles that have a ligand on their surfaces, wherein the phage genome encodes a product of therapeutic genes under the control of a promoter. These and other aspects of the present invention will be apparent with reference to the following detailed description and accompanying drawings. In addition, several references are discussed below which describe in more detail certain procedures and compositions (e.g., plasmids, etc.) and are therefore incorporated by reference in their entirety BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1B are schematic representations of phage vectors for mammalian cell transduction.Figure 1A describes the vector of the mother phage with the wild type plll coat protein.The base vector is the M13 genome with the gene (AmpR) with resistance to ampicillin and the cassette of GFP expression inserted in the intergenic region between pIV and pll (MEGFP3) .The MEGFP3 vector contains the following elements: ori-CMV, SV40 origin of replication and CMV promoter, EGFP, green fluorescent protein gene improved, BGH, and a bovine growth hormone polyadenylation sequence Figure 1B depicts the phage display (MF2 / 1G3) of FGF-plll fusion.

Figure 2 is a graph that shows the number of cells expressing the green fluorescent protein when the FGF phages are incubated with the cells in the presence of increasing amounts of free FGF. Figures 3A and 3B are graphs demonstrating the expression of reporter genes (GFP (3A) and ßGal (3B) in target cells as a function of phage titration Figures 4A-4D are a graphical representation demonstrating specificity of selected FGF2 Figure 5 is a graph showing the number of FGF of positive cells resulting from the incubation of polylysine + phage or FGF-polylysine + phage Figure 6 is an image obtained by scanning a Western blot analysis representing the detection of FG F2-pl 11 fusion protein in protein extracts of purified FGF2 phage (FGF2-MEGFP) Figures 7A and 7B are bar graphs of ELISA detection of FGF2 on phages FGF2. Figure 7A describes the amount of phage protein detected using the empty MEGFP3 vector and the fusion construct of FGF2 (FGF2-MEGFP) Figure 7B describes the amount of FGF2 detected on the phages that the construct has Fusion Fig. 8A and 8B are bar graphs representing the transduction of COS cells by FGF2 phage.

Figures 9A and 9B are fluorescent microscopy images of stable transfectants expressing the GFP reporter gene. DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention is generally directed to methods for the delivery of therapeutic agents in target cells and tissues in a site-specific manner and for constructions and compositions useful in such applications. An application of the description of the invention is gene replacement or gene breeding therapy. In simple terms, the vectors and methods described herein can be used in the treatment of individuals whose tissues lack the ability to produce sufficient quantities of certain important molecules and compounds, e.g., necessary enzymes. Another application of the invention described uses the capacity of vectors described in target cells with improved specificity, when said ideal vectors are formed for the treatment of a variety of tumors and other malignancies. As described in detail herein, preferred vectors for use in the site-directed delivery of therapeutic agents include filamentous phage particles that express one or more preselected ligands on the surface of viral particles, regardless of the form in which they are present. which ligands bind. Therefore, while the binding means of a ligand are covalent via a fusion protein, the selection of the phage vectors of the present invention is capable of delivering the therapeutic nucleotide sequences to the target cells. Filamentous phage particles are particularly useful vectors as described herein, in part because they have native tropism in mammals. Therefore, there is no need to eliminate the native tropism of a phage vector to make it useful in gene therapy applications, as necessary for the most commonly used retroviral vectors. The phage-based vectors of the present invention also have numerous advantages including, for example, the ability to accommodate large loads; the ability to select specific cellular receptors accurately, without damaging healthy cells, thus improving the expression of therapeutic sequences in the target cell. The above features, and others that will be described in more detail below, make the vectors described herein particularly attractive for applications in gene therapy, which can be easily treated to package, transport and deliver the genes that can be expressed as choice for cells, tissues or organs of a patient in need of treatment. As noted above, the vectors and methods described herein are particularly useful in the treatment of disease conditions that are not treated with more "conventional" therapies. I. BACTERIÓFAGOS.

A. Filamentous Bacteriophages Filamentous phages comprise a group of bacteriophages that are capable of infecting a variety of Gram-negative bacteria through interaction with the tip of F pilus. Known filamentous phages include M13, f1 and fd. The genomes of these phages with single-stranded DNA, but replicated through a double-stranded form. The phage particles are assembled in the bacteria and extruded in the medium. Because bacteria continue to grow and divide, although at a slower rate than uninfected cells, relatively high phage titers are obtained. In addition, replication and assembly do not seem to be affected by the size of the genome. As a consequence of its structure and life cycle, filamentous phages become a valuable addition in the tool group of molecular biology. In addition, the development of filamentous phage systems has led to the development of cloning vectors, called phagemids, that combine the characteristics of plasmids and phages. The phagemids contain an origin of replication and packaging signal of filamentous phages, as well as a replication plasmid origin. Other elements that are useful for cloning and / or expressing the above nucleic acid molecules are generally present. Such elements include, without limitation, acceptable genes, multiple cloning sites, primer sequences. Phagemids can replicate like other plasmids and can be packaged in phage particles to be rescued by ancillary filamentous phage. As used herein, "filamentous phage particles" refers to particles that contain a phage genome or a phagemid genome. The particles may contain other molecules in addition to the filamentous capsid proteins. Filamentous phages have been developed as a system for displaying proteins and peptides on the surface of the phage particle. By inserting the amino acid molecules into the genes for phage capsid proteins, fusion proteins assembled in the capsids were produced (Smith, Science 228: 1315, 1985; Patent of E.U.A. No. 5,223,409). As a result, the foreign protein or peptide is displayed on the surface of the phage particle. Methods and techniques for displaying phage are well known in the art (see also, Kay and others, Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, 1996). B. Vectors Filamentous phage vectors genius fall into two categories: phage and phagemid genomes. Any type of vector that can be used within the context of the present invention, preferably phage genome vectors were used. Many of the commercial vectors are available. For example, the vector series pEGFP (Clontech; Palo Alto, CA), M13mp vectors (Pharmacia Biotech, Sweden); pCANTAB 5E (Pharmacia Biotech), pBluescript series (Stratagene Cloning Systems, La Jolla, CA) and others that may be used. A particularly useful commercial vector is pEGFP-N1, which contains a green fluorescent protein (GFP) gene under the control of the immediate early CMV promoter. This plasmid also includes an SV40 origin of replication to improve gene expression by allowing replication of the plasmid at high copy number in cells forming the SV40 T antigen. Other vectors are available in the scientific community (see, eg, Smith, in Vectors: A Survey of Molecular Cloning Vectors and their Uses, Rodríguez and Denhardt, eds., Butterworth, Boston, pp. 61-84, 1988) or they can be constructed using normal methods (Sambrook et al., Molecular Biology: A Laboratory Approach, Cold Springer Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publisching, NY, 1995) guided by the principles described below. To a minimum, for use in the present invention, the vector can accept a cassette containing a promoter and a therapeutic gene (transgene) in an operative ligature. Any promoter that is active in the cells that will be transfected can be used. The vector may also have a replication phage origin and a packaging signal to assemble the vector DNA with capsid proteins. Other elements can be incorporated into the construction. In preferred embodiments, the construct includes the transcription terminator sequence, including a polyadenylation sequence, splice donor and acceptor sites as well as an enhancer. Other useful elements for the expression and maintenance of the construct in mammalian cells or other eukaryotic cells can also be incorporated (eg, origin of replication). Because the constructions are conveniently produced in bacterial cells, the elements that are necessary to improve the propagation in the bacteria are incorporated. These elements include an origin of replication, selectable marker and the like (see discussion below). The promoter that controls the expression of the transgene could be active or activatable in the target cell. Within the present invention, the selected cell can be of mammals, birds, plants and the like. Most of the applications of the present invention could involve the transfection of mammalian cells, including humans, canines, felines, equines and the like. The choice of the promoter will depend in part on the type of cell selected and the degree or type of control desired. Promoters that are suitable within the context of the present invention include, without limitation, constitutive and inducible specific tissue, specific cellular type, specific temporal or event specific. Examples of constitutive or non-specific promoters include the SV40 early promoter (U.S. Patent No. 5,118,627), the SV40 late promoter (U.S. Patent No. ,118,627), the early CMV gene promoter (U.S. Patent No. 5,168,062), the bovine papilloma virus promoter and the adenovirus promoter. In addition for viral promoters, cellular promoters are also treated within the context of this invention. In particular, cellular promoters are useful for domestocal genes, so-called, (e.g., p-actin). Viral promoters are generally stronger promoters than cellular promoters. Tissue-specific promoters, particularly, are useful for expression in endothelial cells. Using one of this class of promoters, an extra margin of specificity can be obtained. For example, specific endothelial promoters are especially useful in targeting proliferative diseases involving endothelial cells. Examples of tissue- or cell-specific promoters include specificity for lymphoid cells, specificity for fibroblast cells, specificity for liver, specificity for kidney, specificity for hepatocytes, and the like. Inducible promoters can also be used. These promoters include MMTV LTR (PCT WO 91/13160) inducible by dexamethasone, metallothionine, inducible by heavy metals and promoters with cAMP response elements, inducible by cAMP, heat shock, promoter. Using an inducible promoter, the nucleic acid can be delivered in a cell and can remain inactive until the inducer is added.

This allows for additional control over the production time control of the gene product. The promoters specific to the type of event are activated or regulated only by the presence of an event, such as tumorigenicity or viral infection. The HIV LTR is a well-known example of a specific promoter for events. The promoter is inactive unless the tat gene product is present, which occurs due to viral infection, some promoters of the event type are also tissue-specific. Additionally, promoters that are regulated in a coordinated manner with a particular cellular gene can be used. For example, promoters of genes that are coordinately expressed when a particular FGF receptor gene is expressed can be used. Then, the nucleic acid can be transcribed when the FGF receptor is expressed, such as FGFR1, and not when FGFR2 is expressed. This type of promoter is especially useful when the pattern of expression of the FGF receptor in a particular tissue is known, so that specific cells within the tissue can be removed by the transcription of a gene of the cytotoxic agent without affecting the surrounding tissues. Examples of promoters described herein include promoters for alpha-fetoprotein, alpha-actin, myo D, carcinoembryonic antigen, VEGF receptor (Access to Gene Bank No. X89776); FGF receiver; TEK or union 2 (Access to Gene Bank No. L06139); union (Access to Bank of Genes Nos. X60954; S89716); Urokinase receptor (Access to Gene Bank No. S78532); selectins E and P (Access to Gene Bank Nos. M64485; L01874); VCAM-1 (Access to Gene Bank No. M92431); endoglina (Access to Gene Bank No. HSENDOG); endosialin (Rettig et al., PNAS 89: 10832, 1991); alpha integrin V-beta 3 (Villa-García et al., Blood 3: 668, 1994; Donahue et al., BBA 1219: 228, 1994); Endothelin-1 (Access to Gene Bank Nos. M25377 J04819; J05489 (; ICAM-3 (Access to Gene Bank No. S50015); E9 antigen (Wang et al., Int. J. Cancer 54: 363, 1993) von Willebrand factor (Genes Bank Nos. HUMVWFI HUMVWFA), CD44 (Access to Gene Bank No. No. HUMCD44B) CD40 (Access to Gene Bank Nos. HACD40L; HSCD405FR) vascular-endothelial cadherin (Martin-Padura et al. , J. Pathol 775: 51, 1995), slot 4 (Uyttendaele et al., Development 722: 2251 1996) antigen associated with high molecular weight melanoma prostate-specific antigen-1, probasin, FGF receptor VEGF receptor, erb, B2 , erb B3, erb B4; MUC-1; HSP-27; int-1; int-2 CEA, HBEGF receptor, EGF receptor, tyrosinase, MAGE, IL-2 IL-2 receptor, prostatic acid phosphate, probasin, prostate-specific membrane antigen, alpha-crystalline, EGFR, PDGF receptor, integrin receptor, -actin, heavy myosin chains of SM1 and SM2, calponin-h1, angiotensin alpha receptor SM22, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, immunoglobulin heavy chain, light chain . * .-. < * ~ A, 'Í.k- 8,? immunoglobulin, CD4, and the like are useful within the context of this invention. In addition to the promoter, repressor sequences, negative regulators or tissue-specific silencers can be inserted to reduce non-specific expression of the cytocid or prodrug. Multiple repressor elements can be inserted into the promoter region. The repression of the transcription is independent in the orientation of the repressor elements or distance of the promoter. A type of repressor sequence is an isolated sequence. Such sequences inhibit transcription (Dunaway et al., Mol Cell Biol 77: 182-9, 1997; Gdula et al., Proc Nati Acad Sci USA 93: 9378-83, 1996, Chan et al., J Virol 70: 5312-28, 1996; Scott and Geyer, EMBO J 14: 6258-67, 1995; Kalos and Fournier, Mol Cell Biol 15: 198-207, 1995; Chung et al., Cell 74: 504-14, 1993) and will silence background transcription. . The negative regulatory elements have been characterized in the promoter regions of a number of different genes. The functions of repressor elements such as a transcription repressor in the absence of factors, such as steroids, such as NSE in the promoter region of the ovalbumin gene (Haecker et al., Mol.Endocrinology 9: 1113-1126, 1995). These negative regulatory elements bind to oviduct-specific protein complexes, none of which are sensitive to steroids. Three different elements are located in the ovalbumin gene promoter. The oligonucleotides corresponding to the portions of these elements represent the viral transcription of the TK reporter. One of the silencing elements forms the sequence identity with the silencers in other genes (TCTCTCCNA). The repressor elements have been identified in the promoter region of the collagen II gene. The gel delay studies show that the nuclear factors of HeLa cells bind specifically to the DNA fragments that contain the silencing region, while the nuclear extracts of chondrocytes do not show any binding activity (Savenger et al., J. Biol. Chem. 265 (12): 6669-6674, 1990). It has been shown that repressor elements regulate transcription in the carbamyl phosphate synthetase genes (Goping et al., Nucleic Acid Research 23 (10): 1717-1721, 1995). This gene is expressed only in two different types of cells, hepatocytes and epithelial cells of the intestinal mucosa. Negative regulatory regions have been identified in the promoter region of the acetyltransferase choline gene, the albumin promoter (Hu et al., J. Cell Growth Differ., 3 (9): 577-588, 1992), the phosphoglycerate gene promoter. kinase (PGK-2) (Misuno et al., Gen 779 (2): 293-297, 1992) and in the 6-phosphofructo-2-kinase / fructose-2,6-bisphosphatase gene, in which the regulatory element negative inhibits transcription in non-hepatic cell lines (Lemaigre et al., Mol Cell Biol. 77 (2): 1099-1106). In addition, the negative regulatory element Tse-1 has been identified in a number of liver-specific genes, including tyrosine aminotransferase (TAT). The expression of the TAT gene is specific to the liver and inducible by glucocorticoids and the signal path of cAMP. It has been shown that the cAMP response element (CRE) selects for repression by the elements specific for hepatocytes (Boshart et al., Cell 67 (5): 905-916, 1990). In the preferred embodiments, the elements that increase the expression of the desired product are incorporated into the construction. Such elements include the internal ribosome binding sites (IRES, Wang and Siddiqui, Curr Top, Microbiol, Immunol 203: 99, 1995, Ehrenfel and Semler, Curr Top, Microbiol, Immunol 203: 65, 1995, Rees and others, Bioechniques 20: 102, 1996; Sugimoto et al., Biotechnology 72: 694, 1994). IRES increases the efficiency of translation. As well as, other sequences can improve expression. For some genes, the sequences, especially at the 5 'end, inhibit transcription and / or translation. These sequences are usually palindromas that can form hairpin structures. Any of the sequences in the nucleic acid can generally be delivered in suppressed form. The expression levels of the transcription or translation product were analyzed to confirm or to ascertain that the sequences affect expression. The transcription levels can be analyzed by any known method, including Northern blot analysis, protection of the Rnas probe and the like. Protein levels can be analyzed by any known method including ELISA. In preferred embodiments, the phage has a replication origin suitable for transfected cells. Five viral replication systems can be used, such as ori EBV and the EBNA gene, SV40 ori and the T antigen, or ori BPV. Other mammalian replication systems can be exchanged. Also, replication genes can cause a high number of copies. The expression of the therapeutic genes of the phage genome can be improved increased the number of copies of the phage genome. In one method, the SV40 origin of the replication is used in the presence of the SV40 T antigen to produce several hundred thousand copy numbers. The T antigen gene can be present around in the cells and is introduced separately, or is included in the genome of phage under the transcriptional control of a suitable cellular promoter. Other viral replication systems increase the number of copies that can also be used, such as the origin of EBV and EBNA. In other preferred embodiments, the peptides or other portions that allow or promote the escape of the vectors (and any molecule bound to or encompassed in the present) from the endosome, are incorporated and expressed on the surface of the bacteriophages. Said "different portions" include molecules that by themselves are peptides that have the ability to damage the membrane endosomal, facilitating the escape of the vector, and the molecules that on the other hand they mimic the endosomal escape properties of the peptide sequences described herein (see, for example, Published PCT Application No. WO96 / 10038 and Wagner et al., PNAS 89: 7934-7938, 1992, the descriptions of which are incorporated herein by reference). The peptide sequences conferring the escape ability of the endosome are particularly preferred. Such sequences are well known and can be easily covalently or genetically fused into a coat protein, such as the III gene or the VIII gene of filamentous phages. Although fusion of one or more peptide sequences in a coat protein is described herein as a preferred embodiment, it should be understood that different methods of binding and other portions of peptides are useful as described herein. Therefore, an example of filamentous phage exhibited twice has a ligand (e.g., FGF) as a fusion for the III gene and an endosomal escape peptide fused to gene VIII. Ligand locations and escape sequences are interchangeable and, therefore, may reside in the same fusion protein. Exhaust sequences that are suitable include, without limitation, the following illustrative sequences: a Pseudomonas exotoxin peptide (Donnelly, J, J., Et al., PNAS, 90, 3530-3534, 1993); influenza peptides such as HA peptide and peptides derived therefrom, such as FP13 peptide; the fusogenic peptide of the Sendai Virus; the fusogenic sequence of the HIV gpl protein, the fusogenic peptide of Paradaxin; and the fusogenic peptide of Meilittin (see published PCT Publication No. WO96 / 41606, the disclosure of which is incorporated herein by reference). Examples of two other endosome disruptive peptides, which are sometimes called fusogenic peptides, are: GLFEAIEGFIENGWEGMIDGGGC (SEQ ID No. 1); and GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID No. 2). Other peptides useful for disruption endosomes can be identified by several general characteristics. For example, endosome disruption peptides are approximately 25-30 residues in length, containing an alternate pattern of hydrophobic domains and acidic domains, and a low pH (e.g., pH 5) to form a-helices unfriendly. The endosome disruptive peptide may be present as one or multiple copies at the N or C terminus of the ligand. The escape peptides are also selected using a molecular evolution strategy. In summary, in one strategy, a bank of random peptides is treated in the gene VIII protein of a phage vector that has a ligand fused to the III gene and that carries a detectable (eg, GFP) or selectable marker (e.g., drug resistance). Mammalian cells are infected with the bank and selected cells by marker detection. Cells that have more efficient endosomal escape could have higher expression or greater resistance. Multiple cycles of selection can be carried out to reduce the complexity of the recovered peptides encoding the genes. The peptide genes were recovered and treated in the phage vectors. further, or alternatively, membrane disruptive peptides can be incorporated into the complexes. For example, adenoviruses are known to improve disruption of endosomes. Virus-free viral proteins, such as HA-2 haemagglutinin from influenza virus, also impair endosomes and are useful in the present invention. Other proteins can be tested in the analyzes described here to find specific agents that deteriorate the endosome which improves the supply of genes. In general, these proteins and peptides are antipathetic (see Wagner et al., Adv. Drug, Del. Rev. 14: 113-135, 1994). Another sequence that can be included in a vector is a sequence that facilitates the circulation proteins in the nucleus. For improved expression, it is preferred that the therapeutic nucleotide sequence circulate in the nucleus. This nuclear translocation, so-called, or nuclear localization sequences (NLS), are generally rich in positively charged amino acids because the carboxyl terminus of the gene VIII protein of filamentous phages already has a positive charge, increased loading and the probability of nuclear transport can be increased by fusing the NLS sequences of known mammalian cells with the gene VIII protein. NLS fusions with other coat proteins of filamentous phages can be substituted. The NLS can be fused with a capsid protein other than a ligand / capsid fusion. Examples of NLS sequences include those that resemble the short basic NLS of the SV40 T antigen; the nucleoplasmin bipartite NLS; the A1 sequence of ribonucleoprotein; the small nuclear ribonucleoprotein U1A sequence and the T-lymphocyte virus-1Tax protein. Other useful NLS sequences include the NLS HIV matrix protein; and nuclear translocation components, importaina / hSRPI and Ran / TC4; the generalized sequence KXX (K / R) flanked by Pro or Ala; the nuclear translocation sequence of nucleoplasmin; or the antennapedia NLS (see, WO 96/41606). In some cases, the nucleic acid that condenses the peptides is linked to non-basic nuclear localization sequences that function to transport the nucleoprotein. Examples of such sequences include influenza nucleoprotein and HIV MA protein. Other useful NLSs include the A1 protein of hnRNP, a protein that carries the ribonucleoprotein complexes. Other useful NLS are included in peptides such as NBC1 and NBC2, whose function also condenses the nucleic acid (See, WO 96/41606). The present invention therefore contemplates the use of the above sequences as well as others described herein. A random peptide bank of the sequences can be screened for novel sequences that promote nuclear translocation. In summary, in one of these methods, a random peptide gene bank is fused to the filamentous phage genes VIII, VII or IX and screened for efficient nuclear translocation by analyzing the infected cells for the expression of the reporter or selection gene. of the drug Additional factors that enhance the expression of the transgene can be included. Such factors can be identified by a method in which the sequences are fused to phage coat proteins and the phages are selected based on the expression of the efficient reporter gene. The candidate sequences are the DNA repair enzymes or polymerases of mammalian cells or single-stranded DNA viruses. Phage presenting the ligand as a fusion with a phage coat protein are treated to contain the appropriate coding regions. For filamentous phages, genes III and VIII are usually used. Other capsid proteins can be substituted. Techniques for inserting the ligand encoding the sequence into a phage gene are well known (see, for example Sambrook et al., Supra, Ausubel et al. Supra). In certain embodiments, the propagation or stable maintenance of the construct may be convenient or may be necessary to bind a sufficient amount or concentration of the gene product for effective gene therapy. Examples of replication and stable eukaryotic origins of replication are known.

II. LIGANDS As used herein, "ligand" refers to any peptide, polypeptide or protein or non-protein, such as an imitation peptide, that is capable of binding to a cell surface molecule and entering. Internment through the endosomal route is a preferred mode of entry. As used herein, "binding to a receptor" refers to the ability of a ligand to specifically recognize and bind detectably to a receptor, as analyzed by normal in vitro assays Within the context of this invention, the ligand is conjugated to a protein from a bacteriophage, either as a fusion protein or through chemical conjugation, and used to deliver the loading of nucleic acid (ie therapeutic genes) into a cell Numerous molecules are known to bind to a specific receptor and they are interned, generally by means of endosomes, Still other molecules are antibodies and antibody derivatives, In addition, methods are provided for the selection of other ligands that satisfy the criteria presented above The fragments of these ligands can be used within the present invention , along the fragment that retains the binding capacity in the appropriate cell surface molecule. ligands can be used with substitutions or other alterations, but they retain the binding capacity. Also, a particular ligand refers to a polypeptide having an amino acid sequence of the native ligand, as well as the modified sequences (e.g., having substitutions, deletions, insertions or additions of amino acids compared to the native protein) while the Ligand retains the ability to bind to its receptor on an endothelial cell and is internalized. Ligands also encompass muteins that have the ability to bind to their receptor that expresses cells and internalizes. Such muteins include, but are not limited to, those produced by replacing one or more of the cysteines with serine as described herein. Normally, said muteins have conservative amino acid changes, the DNA encoding the muteins could be modified by replacing the degenerative codons, hybridizing under conditions of at least low restriction to the native DNA sequence encoding the wild-type ligand. The DNA encoding a ligand can be prepared synthetically based on the amino acid sequence or known DNA, isolated using methods known to those skilled in the art (e.g., PCR amplification), or obtained from commercial sources or other sources. The DNA encoding the ligand can differ from the previous sequences by substituting degenerative codons or encoding different amino acids. Differences in amino acid sequences such as those present between the homologous ligand of different species as well as between individual organisms or species are tolerated while the ligand binds to its receptor. Ligands can be isolated from natural sources or can be synthetic, such as recombinant media or chemical synthesis. It is not necessary that the ligands used in the context of this invention retain any of the biological activities in vivo, other than the binding of a receptor on a cell and can be internalized. However, it may be convenient in certain contexts for a ligand to manifest certain of its biological activities. For example, if VEGF is used as a carrier for DNA encoding a molecule useful in wound healing, it might be convenient for VEGF to exhibit the permeability activity of the vessel and the promotion of migration of fibroblasts and angiogenesis. In the examples, FGF muteins with reduced mutagenic activity have been constructed by site-directed mutagenesis. With or without reduced mutagenic proteins can also be constructed by exchanging the receptor binding domain with the receptor binding domain of a related protein. As an example, the FGF2 domain can be interchanged with the binding domain of the FGF7 receptor to create an FGF that does not cause proliferation and can accelerate the binding profile. If the ligand has been modified so that it lacks one or more biological activities, the binding and internalization can be easily analyzed by any of the following tests or equivalent tests. Generally, these tests involve labeling the ligand, incubating them with target cells, and visualizing or measuring the intracellular label. For example, in summary, the ligand can be : * J í j > SifcjkiÍ.i, .. fluorescently labeled with FITIC or radiolabelled with 125 μl, incubated with cells and examined microscopically by fluorescence microscopy or confocal microscopy for internment. It will be evident that the teachings provided within the objective of the application whose biological activities are convenient to be maintained. A. Proteins that bind to cells and internalize Ligands can be produced by recombinant means or other means in preparation for binding to phage capsid proteins. The DNA sequences and methods for obtaining the sequences of these ligands are well known (see Bank of Genes). Based on the DNA sequences, genes can be synthesized either synthetically (for small proteins), amplified from genomic cells or cDNAs, isolated from genomic libraries or cDNAs and the like. Restriction sites to facilitate cloning in phage or phagemid vector can be incorporated, such as primers for amplification. Molecules include, without limitation, proteins that bind cancer cells, endothelial cells, cardiovascular cells, cells in the eye and the like. Such ligands include growth factors and cytokines. Many developmental factors and families of developmental factors form structural and functional aspects and can be used in the present invention. The families of developmental factors include FGF-1 fibroblast development factors through FGF-15, and vascular endothelial growth factor (VEGF). Other developmental factors, such as PDGF (platelet derived development factor), TGF-a (transforming development factor); TGF-β, HB-EGF, angiotensin, bombesis, erythropoietin, root cell factor. M-CSF, G-CSF, GM-CSF, and endoglin also bind to specific identified receptors on cell surfaces and can be used in the present invention. Cytokines, including interleukins, CSF (colony stimulating factors) and interferons, have specific receptors and can be used as described herein. For example, ligands and ligand / receptor pairs include the urokinase / urokinase receptor (Access to Gene Bank Nos. X02760 / X74309); α-1,3 fucosyl transferase, a1-antitrypsin / E-selectin (Access to Gene Bank Nos. M98825, D38257 / M87862); P-selectin glycoprotein ligand, Selectin ligand P / Selectina P (Access to Gene Bank Nos. U25955, U02297 / L23088), VCAM1 / VLA-4 (Access to Gene Bank Nos. X53051 / X16983); E9 antigen (Blann et al., Atherosclerosis 720: 221, 1996) / TFGß receptor; Fibronectin (Access to Gene Bank No. X02761), a1-collagen type I (Access to the Gene Bank) No. Z74615), ß1-collagen type I (Access to Gene Bank No. Z74616), hyaluronic acid / CD44 (Access to Gene Bank No. M59040); ligand CD40 (Access to Gene Bank No. L07414) / CD40 (Access to Gene Bank No. M83312); Linked EFL-3, LERTK-2 (Access to the Bank of Genes Nos. L37361, U09304) ^ '^ ia ^ mBíA & s ^ ti ^ éSi ^ m, for elk-1 (Access to Gene Bank No. M25269); VE-cadherin (Access to Gene Bank No. X79981); ligand of catenins; ICAM-3 (Access to Gene Bank No. X69819) ligand for LFA-1, and von Willebrand Factor (Access to Gene Bank No. X04385), fibrinogen and fibronectin (Access to Gene Bank No. X92461) ligands for the integrin avß3 (Access to the Bank of Genes Nos. U07375, L28832). Other ligands include CSF-1 (Access to Gene Bank Nos. M11038, M37435); GM-CSF 8 Access to Gene Bank No. X03021); IFN-a (interferon) (Access to Gene Bank No. A02076; WO 8602862-A); IFN-? (Access to Gene Bank No. A02137; WO 8502624-A); IL-1-a (interleukin 1 alpha) Access to Gene Bank No. X02531, M15329); IL-1-ß (interleukin 1 beta) Access to Gene Bank No. X02532, M15330, M15840); IL-1 (Access to Gene Bank No. K02770, M54933, M38756); IL-2 8 Access to Gene Bank No. A14844, A21785, X00695, X00200, X00201, X00202); IL-3 (Access to Gene Bank No. M14743, M20137); IL-4 (Access to Gene Bank No. M13982); IL-5 (Access to Gene Bank No. X04688, J3478); IL-6 (Access to Gene Bank No. Y00081, X04602, M54894, M38669, M14584); IL-7 (Access to Gene Bank No. J04156), IL-8 (Access to Gene Bank No. Z11685); IL-10 (Access to Gene Bank No. X78437, M57627); IL-11 (Access to Gene Bank No. M57765 M37006); IL-13 (Access to Gene Bank No. X69079, U10307), TNF-a (tumor necrosis factor) (Access to Gene Bank No. A21522); TNF-ß (Access to Gene Bank No. D12614); and ligand GP30 (S58256) for erbB2 Still other ligands include PDGF (Access to Gene Bank No. X03795, X02811), angiotensin (Access to Gene Bank No. K02215) and peptides containing RGD and proteins, such as ICAM -1 (Access to Gene Bank No. X06990) and VCAM-1 (Access to Gene Bank No. X53051) that bind integrin receptors. Other ligands include TNFa (Access to Gene Bank No. A21522, X01394), IFN-? (Access to Gene Bank No. A11033, A11034); IGF-1 (Access to Gene Bank No. A29117, X56773, S61841, X56774, S61860), IGF-II (Access to Gene Bank No. A00734, X06159, Y00693), atrial naturiético peptide (Access to the Bank of Genes No . X54669), endothelin-1 (Access to Gene Bank No. Y00749), coagulation factor Xa (Access to Gene Bank No. L00395, L00396, L29433, N00045, M14327), TGF-ß1 (Access to Gene Bank No. A13751); IL-1a (Access to Gene Bank No. X03833), IL-1β (Access to Gene Bank No. M15330) and endoglin (Access to Gene Bank No. X72012). The family of FGF proteins currently includes FGF-1 (FGF acid or aFGF), FGF-2 (basic FGF or bFGF), FGF-3 (int-2), FGF-4 (hst-1 / K-FGF), FGF-5, FGF-6 (hst-2), FGF-7 (keratinocyte growth factor or KGF), FGF-8, FGF-9, FGF-11 (WO 96/39507), FGF13 (WO 96/39508 , FGF-14 (WO 96/39506) and FGF-15 (WO 96/39509) Other peptides that are reactive with the FGF receptor, ie any peptide that specifically interacts with the FGF receptor, preferably the receptor of affinity FGF, and is transported by means of endosomes in the cell by virtue of their interaction with the FGF receptor which are suitable within the present invention S. Antibodies to receptors that are interned The antibodies to molecules that are expressed on the surface of the cells that are useful within the context of the present invention as large as the antibody that are interned in the next binding .. Such antibodies include, but are not limited to, anti bodies for FGF receptors, VEGF receptors, urokinase receptor, E and P selectins, VCAM-1 receptor, PDGF, TGF receptor, endosialin, alphav beta3 integrin, LFA-1, E9 antigen, CD40, cadherins, and elk-1 . Antibodies that are specific for cell surface molecules expressed by cells that are easily generated as a monoclonal or polyclonal antiserum. Many antibodies are available (eg, from American Type Culture Collection, Rockville, MD). Alternatively, antibodies for ligands that bind / internate can be used. In this strategy, the phage particles will have antibodies on their surface, which will then form complexes with the ligand (see additional discussion below). Within the context of the present invention, the antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., Fab and F (ab ') 2, Fv variable regions, or regions of ... i.¡ »..3M3feaafac < . > complementary determination). The antibodies are generally accepted as specific against indolicidin analogs and bind with Kd of greater than or equal to 10"7M, preferably greater than or equal to 108 M. The affinity of a monoclonal antibody or binding standard can be easily determined by a someone with ordinary experience in the matter (see Scatchard, Ann. NY Acad. Sci. 57: 660-672, 1949) Once the appropriate antibodies have been obtained, they can be isolated or purified by many techniques well known to those with ordinary experience in the art Commercially available antibodies to cell surface molecules can be used if they are internalized In summary, the antibodies are raised by immunizing mice, rats, rabbits, or other animals with normal, tumorigenic or cultured cells. Various immunization protocols can be found, for example, in Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratoy, 1988 and Coligan I Current Protocols in Immunology, Greene Publishing, 1995. After immunization, the spleen and lymph nodes were recovered to generate hybridomas or the serum is recovered for polyclonal antibodies. Hybridomas are preferred (see, U.S. Patent Nos. 32,011, 4,902,614, 4,543,439 and 4,411,993, Harlow and Lane, supra; an Coligan et al., Supra, for protocols). Hybridomas secreting antibodies were developed, and the antibodies were probed to bind to the immunization cells by ELISA, manca section, flow cytometry, confocal microscopy and the like.

The positive antibodies were also tested for internment. An analysis that is used is a test for an antibody that kills cells. Briefly, the hybridoma antibody test and the cells tested were incubated. The unbound antibody was washed. A second antibody stage, such as an anti-IgG antibody, conjugated to saporin was incubated with the cells tested. Dead cells were secured by any known assay, including trypan blue exclusion, MTT uptake, fluorescein diacetate tinsion and the like. Other techniques can also be used for the construction of monoclonal antibodies (see Huse et al., Science 246X1275-1281, 1989, Sastry et al., Proc. Nati, Acad. Sci. USA 86: 5728-5732, 1989, Alting-Mees and others. , Strategies in Molecular Biology 3.J-9, 1990, describing recombinant techniques). These techniques include the cloning of heavy and light chain immunoglobulin cDNA into suitable vectors, such as? LmmunoZap (H) and? LmmunoZap (L). These recombinants were also individually protected or co-expressed to form the Fab fragments or antibodies (see Huse et al., Supra; Sastry et al., Supra). The positive plaques can subsequently be converted into a non-lytic plasmid allowing the higher level expression of the E. coli monoclonal antibody fragments. Similarly, portions of fragments, such as Fab and Fv fragments, of antibodies can be constructed using conventional enzymatic digestion or recombinant DNA techniques to give isolated variable regions of an antibody. Within one embodiment, the genes encoding the variable region of a hybrid that produces a monoclonal antibody of interest are amplified using variable region nucleotide primers, in addition, the techniques can be used to change a "murine" antibody to an antibody of "human beings", without altering the binding specificity of the antibody. C. Selection of different ligands Other receptor binding ligands can be used in the present invention. Any protein, peptide, analog, peptide mimic or fragments thereof that bind to a cell surface receptor and internate can be used. These ligands can be identified and selected by a method such as a phage display (see, for example, U.S. Patent No. 5,223,409); and the co-pending request "Methods using phage display for selecting internalizing ligands for gene delivery", filed on August 29, 1997). In summary, in this method, the DNA sequences are inserted into gene II or gene VIII of filamentous phages, such as M13. Several vectors with multiple cloning sites have been developed for insertion (McLafferty et al., Gen 728: 29-36, 1993; Scott and Smith, Science 249: 386-390, 1990; Smith and Scott, Methods Enzymol., 217: 228- 257, 1993). The inserted DNA sequences can be generated randomly or be variants of a - i. & ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Single-chain antibodies can be easily generated using this method. Generally, the code inserted from 6 to 20 amino acids. The peptide encoded by the inserted sequence is displayed on the surface of bacteriophages. Bacteriophages expressing a binding domain for the target cells are selected for the binding of the cells or preferably for the expression of a detectable or selectable transgene encoded by the bacteriophage genome. For the methods of selection or detection of transgenes, the cells were developed in selective medium (eg, containing the drug) or isolated on the basis of expression (e.g., flow cytometry). The inserted DNA was recovered, generally by the amplification of lysed cells, analyzed (e.g., DNA sequence analysis) and used in the present invention (Barry et al., Supra). The eluted phages were propagated in the host bacterium. In any of these methods, additional selection cycles can be carried out to choose a binding of some phages with high affinity. The DNA sequence of the insert in the binding phage was then determined. Once the described amino acid sequence of the binding peptide is known, sufficient peptide for use in chemical conjugates can be made by recombinant means or synthetically and by recombinant means to be used as a fusion protein. The peptide can be generated as a random selection of two or more peptides, in order to maximize affinity or binding. D. Modifications of the internal receptor binding ligands The ligands for use herein will be adapted for a particular application. The means for the modification proteins are provided below. In summary, additions, substitutions and deletions of amino acids can be produced by any commonly used recombinant DNA method. The encoded peptides, especially those lacking in function, and chemical peptides, which retain their specific binding and internment activities, are also contemplated in the use of the present. The modifications also include the addition or deletion of residues, so that the addition of a cysteine facilitates the conjugation and form of conjugates containing a defined molar ratio (e.g., 1: 1) of the polypeptides (see, for example U.S. Patent No. 5,175,147; PCT Application No. WO 89/00198, U.S. Patent Series No. 07 / 070,797; PCT Application No. WO 91/15229; and U.S. Patent Series No. 07 / 505,124) . Still other useful modifications include adding the sequence that undergoes post-translational modification (eg, myristylation, palmatylation, phosphorylation, ribosylation) that enhance or alter protein function, stability or the like. The modification of the polypeptide can be affected by any means known to those with experience in this field. Preferred methods herein rely on modification of the DNA encoding the polypeptide and expression of the modified DNA. DNA encoding one of the receptor binding ligands described above can be mutagenized using standard methodologies. For example, cysteine residues that are responsible for the formation of aggregates that can be deleted or replaced. If necessary, the identity of cysteine residues that contribute to the formation of aggregates can be determined empirically, by the deletion and / or replacement of a cysteine residue and the determination of the resulting protein aggregates in solutions containing regulatory solutions and salts physiologically acceptable. In addition, fragments of these internment ligands that bind to the receptor can be constructed and used. The binding region of most of these ligands have been delineated. For example, the binding region of the FGF2 receptor has been identified by mutation analysis and the agonists / antagonists of the FGF peptide to reside between residues 33-77 and between 102-129 of the form form of amino acid 155 (Baird and others, PNAS 85: 2324; Erickson et al., Biochem 88: 3441): Exons 1-4 of VEGF are required for receptor binding. The fragments are also shown to be joined and internalized by any of the tests described herein. Mutations can be made by any method known to those skilled in the art, including site-specific mutagenesis or site-directed mutagenesis of DNA encoding the protein and use of DNA amplification methods using primers to introduce and amplify alterations in the DNA pattern, such as the CPR assembly overlapping the extension (SOE). Site-directed mutagenesis is usually performed using a phage vector that has single-stranded or double-stranded forms, such as the M13 phage vectors, which are well known and commercially available. Other suitable vectors containing a phage origin from a single strand of replication can be used (See, for example, Veira et al., Meth, Enzymol 75: 3, 1987). In general, site-directed mutagenesis is carried out by preparing a single-stranded vector encoding the protein of interest (ie, a member of the FGF family or a cytotoxic molecule, such as saporin). An oligonucleotide primer containing the desired mutation within a region of homology to the DNA in the single stranded vector is annealed to the vector followed by the addition of a DNA polymerase, such as polymerase I of E. DNA. coli (Klenow fragment), which uses the double-stranded region as an initiator to produce a heteroduple in which one thread encodes the altered sequence and the other original sequence. The heteroduple is introduced into the appropriate bacterial cells and the clones that include the desired mutation are selected. The resulting altered DNA molecules can be expressed recombinantly in appropriate host cells to produce the modified protein.

Suitable conservative amino acid substitutions are well known and can generally be made without altering the biological activity of the resulting molecule. For example, such substitutions are generally formed by the exchange within the groups of polar residues, charged residues, hydrophobic residues, small residues, and the like. If necessary, such substitutions can be determined merely empirically by testing the resulting modified protein for the ability to bind and enter into the binding of the appropriate receptors. Those that retain the capacity are suitable for use in the conjugates and methods of the present. Thus, an amino acid residue of an internalized ligand bound to the receptor is not essential if the polypeptide has been modified by deletion or alteration of the residue that has substantially the same binding capacity in its receptor and is internalized in a bound agent as an unmodified polypeptide. E. Peptide Imitation Ligands Ligands or fragments thereof that bind to a cell surface receptor and enter, but which are imitations of the "true" polypeptides, are also contemplated for use in the present invention. Therefore, in one aspect, the invention contemplates the preparation and use of peptide mimics without peptides useful for mimicking the activity of peptides, which make additional sources of peptide mimic selection ligands that can bind to vectors based on phage of the present invention. Methods for generating and identifying the imitations of useful peptides as described herein are known in the art, (see for example WO 93/17032). For example, the application mentioned above describes a process for preparing mock compound of peptides useful for mimicking the activity of the peptides and describing the peptide-like activity of said imitation. Similarly, the production of the peptide mimic drugs via using chemically modified moieties to mimic the structure of the antibody, based on conformational studies, is described in U.S. Pat. No. 5,331,573. The test methods of the drugs that were prepared are also described herein. Imitations of antibody peptides are therefore useful as described herein, not only as ligands but as useful molecules in the ligation of phage particles for the selection of ligands. Other peptide mimic molecules useful as ligands and / or "ligatures" herein are disclosed in published PCT Publication No. WO 92/20704; Brandt. and other Antimicrob Agents Chemother, 40: 1078, 1996; Sepp-Lorenzino, et al., Cancer Res, 55: 5302, 1995; and Chander et al., J. Pharm Sci, 84: 404, 1995. Although the fact that said mimics are not real peptides, several covalent and non-covalent means for joining said peptide mimics to proteins ^ - "*" f * a cover with phages can be used as described herein. F. Expression Vectors for the Production of Ligands As used herein, "operative ligation" or operative association of two nucleotide sequences refers to the functional relationship between said sequences. Nucleotide sequences include, but are not limited to, DNA encoding a product, DNA encoding a signal sequence, promoters, enhancers, translational stop sites and polyadenylation signals. For example, the operative ligation of DNA encoding a cytocide in a promoter refers to the physical and functional relationship between the DNA and the promoter of said DNA transcription that is initiated from the promoter by an RNA polymerase that specifically recognizes the junctions, and transcribes the DNA. Host organisms include those organisms in which recombinant production of heterologous proteins, such as bacteria (e.g., E. coli), yeast, has been carried out. (for example Saccharomyces cerevisiae and Pichia pastoris), mammalian cells and insect cells. Currently preferred host organisms are E. coli bacterial strains. The constriction of the DNA encoding the desired protein is introduced into the plasmid for expression in an appropriate host.

In preferred embodiments, the host is a bacterial host. The sequence encoding the ligand is preferably the codon optimized for expression in the particular host. Therefore, for example, if FGF-2 from humans is expressed in the bacterium, the codons could be optimized for bacterial use. For the smaller coding regions, the gene can be synthesized as a single oligonucleotide. For larger proteins, the splicing of multiple oligonucleotides, mutagenesis or other techniques known to those skilled in the art was used. The nucleotide sequences in plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription. The sequence of the nucleotides encoding the developmental factor or the chimera of the developmental factor also includes the DNA encoding a secretion signal, resulting in the peptide being a precursor protein. The resulting processed protein can be recovered from the periplasmic space or from the fermentation medium. Plasmids used herein include a promoter in operable association with the DNA encoding the protein or polypeptide of interest and are designed for the expression of proteins in the bacterial host. Suitable promoters for the expression of proteins and polypeptides are widely available and are well known in the art. Inducible promoters or constitutive promoters that bind in the regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-like phage promoters, such as the T3, T5 and SP6 promoters, the trp, Ipp and ^^ j ^^^^^^ J ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^ j ^ gg ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ j ^ lac, such as lacUV5, from E. coli; the P10 gene or polyhedron promoter of baculovirus / insect cell expression systems (see, for example, U.S. Patents Nos. 5,243,041, 4,242,687, 5,266,317, 4,745,051 and 5,169,784) and inducible promoters of other eukaryotic expression systems. For the expression of the proteins of said promoters they are inserted in a plasmid in ligature operative with the control region such as the lac operon. Preferred promoter regions are those that are inducible and functional in E. coli: Examples of suitable inducible promoters and promoter regions include, but are not limited to: the lac E. coli operator responsive to isopropyl β-D-thiogalactopyranoside ( IPTG; see, Nakamura et al., Cell 78: 1109-1117, 1979); the metallothionine promoter of the regulatory elements of multiple heavy metal sensitive (e.g., zinc) induction (see, e.g., U.S. Patent No. 4,870,009 to Evans et al); the T71lac promoter of the phage in IPTG-sensitive (see, for example, U.S. Patent No. 4,952,495; and Studier et al., Meth. Enzymol, 785: 60-89, 1990) and the TAC promoter. The plasmids preferably include a selectable marker gene or genes that are functional in the host. A selectable marker gene includes any gene that confers a phenotype on the bacterium that allows the transformed bacteria to identify and develop selectively from a vast majority of non-transformed cells. Selectable marker genes for host bacteria, for example, include g £ foj ^^^^ £ & tf ^ ¡&& & amp; amp; amp; ampicillin resistance gene (Amp4), the tetracycline resistance gene (Tcr) and the kanamycin resistance gene (Kanr). The kanamycin resistance gene is currently preferred. Plasmids also include DNA encoding a signal for secretion of the operably linked protein. Secretion signals suitable for wide use are available and are well known in the art. Prokaryotic and functional eukaryotic secretion signals in E. coli can be used. Currently preferred secretion signals include, but are not are limited to those that are encoded by the following E. coli genes: ompA, ompT, ompF, ompC, beta-lactamase and alkaline phosphatase and the like (von Heijne, J. Mol. Biol. 784: 99-105, 1985). Using the methods described herein, a person skilled in the art can substitute secretion signals that are functional in yeast, insect or mammalian cells to secrete the proteins of the cells. In preferred embodiments, the DNA plasmids also include a transcription terminator sequence. The complete transcription terminator can be obtained from a gene that encodes the protein, which may be the same or different from the inserted gene or the source of the promoter. Particularly preferred plasmids for transformation of E. coli cells include pET expression vectors (see U.S. Patent No. 4,952,496; of Novagen, Madison, Wl). Such plasmids include pET 11a, which ^^ fe¿ - .. rta ^^^^ aato? ^^ a ^ a contain the T71lac promoter, the T7 terminator, the lac operator of inducible E. coli and the repressor gene of lac; pET 12a-c, which contains the T7 promoter, the T7 terminator and the ompT secretion signal of E. coli; and pET 15b containing a His-Tag ™ leader sequence for use in purification with a His column and a thrombin cleavage site allowing cleavage following purification on the column, the T7-lac promoter region and the T7 terminator . Other preferred plasmids include the pKK plasmids, particularly pKK 223-3, which contains the tac promoter (Pharmacia Biotech). Plasmid pKK has also been modified by replacing the ampicillin resistance gene with a cassette of kanamycin resistance plasmids (Pharmacia Biotech). Other plasmids include the pIN-lllompA plasmids (see U.S. Patent No. 4,575,013), which have a cloning site linked to the transcriptional reading frame with four functional fragments derived from the E. coli lipoprotein gene and an ompA signal sequence. Baculovirus vectors, such as pBlueBac (also called pJVETL and derivatives thereof), particularly pBlueBac III (Invitrogen, San Diego, CA) can be used for the expression of polypeptides in insect cells. A DNA construct can be made in a baculovirus vector and then co-transfected with the wild-type virus into the sf9 insect cells of Spodoptera frugiperda (see, eg, Luckow et al., Bio / technology 6: 57-55, 1988; and U.S. Patent No. 4,745,051).

Expression vectors compatible with eukaryotic cells, preferably those compatible with mammalian cells, can also be used to form recombinant nucleic acid molecules for use in the present invention. Expression vectors of mammalian cells are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing suitable restriction sites for insertion of the desired DNA segment and provide signals that are required for expression genes in a mammalian cell. Normally said vectors are the vectors of the pREP and pEBVhis series available from Invitrogen (San Diego, CA), vectors pTDT1 (ATCC # 31255), pCP1 (ATCC # 37351) and pJ4W (ATCC # 37720) available from American Type Culture Collection (ATCC) and similar. Successively transformed cells, i.e., cells containing a nucleic acid molecule of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of a rDNA of the present invention can be adjusted for analysis to detect the presence of a specific rDNA using a nucleic acid hybridization method such as that described by Southern, J. Mol. Biol. 98: 503, 1975 or Berent et al., Biotech. 3: 208, 1985. Furthermore, by directly analyzing the presence of rDNA, the successive transformation can be confirmed by well-known immunological methods for the presence of the expressed protein. For example, cells transformed successively with an expression vector that produces proteins that can then be analyzed directly by immunological methods or for the presence of the function of the expressed protein. Other suitable mammalian expression vectors are well known and can be obtained easily from a variety of sources. (See, for example, Ausubel et al., 1995; Sambrook et al., Supra; Invitrogen, San Diego, CA; Novagen, Madison, Wl; Pharmacia Biotech; and others). In several preferred embodiments, the DNA fragment replicates in bacterial cells, preferably in E. coli. The preferred DNA fragment also includes a bacterial origin of replication. Preferred bacterial origins of replication include, but are not limited to, the f1 origin and the E1 origins of replication col. Preferred hosts contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the lacUV promoter (see U.S. Patent No. 4,952,496). Such hosts include, but are not limited to, lysogen E. coli strains HMS174 (DE3) pLysS, BL21 (DE3) pLysS, HMS174 (DE4) and BL21 (DE3). Strain BL21 (DE3) is preferred. The DNA fragments provided also contain a gene encoding a repressor protein, which is capable of representing the transcription of a promoter that contains a binding site for the repressor protein. The promoter may not be repressed by altering the physiological conditions of the cell. Preferred repressor proteins include, but are not limited to, the repressor lacl of E. coli sensitive to induction of IPTG, the temperature sensitive to? c1857 and similar. In a preferred embodiment described herein, the "plasmid" is the phagemid pEGFP-N1 (Clontech; Palo Alto, CA), which contains a green fluorescent protein (GFP) under the control of the readily immediate promoter. The CMV promoter is highly active in a wide variety of mammalian cell lines; however, other promoters of mammalian cells can be used. Examples of other useful promoters active in mammalian cells include viral promoters (e.g., LTRs, MMTV LTR, HIV LTR, SV retrovirals easily and late promoters, Bovine Papilloma Virus, BPV) or non-viral inducible promoters (v. .gr., 15 metallothionine, heat shock, promoters of steroid hormone responses). Still other promoters include those that are constitutive (e.g., Beta Actin) or specific tissue (e.g., alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), alpha actin and Myo D). Additional useful promoters are described in other parts of the present. Preferably, the phagemid also includes an SV40 origin of replication to improve gene expression. Other viral replication systems were also used; for example, the origin EBV and EBNA or BPV are useful as described in the present one.

H ^^ g ^ ^ ^. ^^^^^^^^^^^^^^^^^^^^^^ t ^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^ f ^^^^^^^^ l ^^^^^ A strain of E. coli preferred in which phagemids are propagated is the host strain of £. coli DH5aF '. The systems useful according to the present invention can also benefit from the use of the auxiliary bacteriophages. For example, the 5 M13 auxiliary of M13K07 is particularly useful in conjunction with the bacterial strains mentioned above. Methods for preparing phagemid particles are known in the art and can be modified appropriately depending on the system used, such as those skilled in the art. matter could be appreciated. (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989, Rider et al., In Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, 1996). lll. CONSTRUCTION OF THE BACTERIÓFAGOS MODIFIED BY LIGAND A variety of constructs and methods can be easily adapted for use in binding a phage to a ligand for use in the specific selection and delivery of the therapeutic portion or agent. Therefore, the phages present a ligand that is part of a fusion protein, direct chemical bond or sandwich. Illustrative methods for modifying bacteriophages to present ligands are described below. Ligands are killed as described herein by any suitable method, including DNA technology recombinant, isolation from a suitable source, acquisition of ^ ji¿ jí | fc tÉ ^ ^^^^^^^^^^? a commercial sources, or chemical synthesis. Several preferred methods and modifications for ligands that can facilitate ligation between the ligand and the phage protein are described in published PCT Publication No. WO 96/36362. For example, the DNA encoding the polypeptide ligands can be isolated, synthesized or obtained from commercial sources or prepared as described herein. The expression of recombinant polypeptides can be carried out as described herein; and the DNA encoding these polypeptides can be used as starting materials for the methods herein. The DNA can be prepared synthetically based on the amino acid or DNA sequence or can be isolated using methods known to those skilled in the art, such as PCR, bank hybridization probes, and the like or obtained from commercial sources or other sources. A. Generation of fusion proteins The fusion proteins of the present invention preferably comprise a gene encoding all or a portion of the polypeptide that binds to the receptor of a genetically fused ligand (e.g., FGF2) or ligand in the gene encoding the protein of a baceriofagos particle using methods known to those skilled in the art (see, for example, Smith and Scott Meth Enzimol, 277: 228-257, 1993; Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, 1996). Preferably, the phages are filamentous phages; M13 is described herein as an illustrative preferred embodiment. The preparation of a fusion protein comprising the Mll gene of M13 or Vil of the coat protein are described herein. It should be noted that it is preferred that, when the phage vectors are assembled, the fusion proteins coated with the ligand-phage are the predominant species. Generally, the copy number of fusions coated with the ligand-phage in relation to the wild type coat protein can be easily controlled by displaying the fusion in a higher fusion copy number (type 3 or type 8 vectors) or a number of lower copies (vector type 3 + 3 or type 8 + 8). The nucleotide sequences for the ligands are readily available (eg, from Bank of Genes) or can be synthesized or isolated by standard techniques. The coding regions are inserted into the phage vectors using well-known methods. The nucleotide sequences encoding the ligand-phage fusions can also be modified via the insertion of a mammalian reporter gene, in order to verify the binding and internment, as well as the expression of the nucleic acid charge. A reporter gene of illustrative mammals is EGFP; others include the sequences for β-galactosidase, luciferase, human development hormone (HGH), and secreted alkaline phosphatase. The methods for preparing and using said sequences are described herein and are known to the . fc, * * & . experts in the field (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989). In several preferred embodiments, the expression cassette 5 also includes an origin of replication. SV40 ori is particularly useful for generating high numbers of replication copies in cell lines containing the T antigen (e.g., COS cells). A variety of useful methods and protocols are available which employ these and other origins of replication 10 (see, for example, Sambrook et al., Id). Protein fusions of coatings with ligand can be tested for binding of phage in the relevant receptor (e.g., an analog receptor for the ligand) via known methods such as ELISA. The binding and internment of the fusion was easily analyzed via methods recognized in the art such as immunohistochemistry (Barry et al., Nature Med. 2: 299-305, 1996; Li, Nature Biotech, 75: 559-563, 1997). The modified phage can also be tested for the transduction of the reporter gene in the cells, preferably the mammalian cells, by adding the phages in the cell cultures for a predetermined amount of time; depending on the protocol and reporter used, the number of cells expressing the reporter gene product at subsequent and predetermined time were easily analyzed using known methods. For example, if the reporter gene in a subsequent predetermined time is aa &afea? MÉaaasafeaSagia .. -g_a s-- - * •. < - - - m. »> . «.« -. - a-á -. they evaluate easily using well-known methods. For example, if the reporter gene is EGFP, it moves into the green fluorescent protein (GFP) when the gene's release and expression occurs, or the number of self-fluorescent cells can easily be measured for several additional hours. The results were easily quantified. For example, if fluorescent reporters were used, flow cytometry is particularly useful to measure the distribution of cells expressing the fluorescent reporter gene in the population and by fluorometry of cell extracts to measure the total amounts of reporter protein expressed. A variation of the aforementioned method involves the constriction of a fusion protein including all or a portion of an antibody molecule and a phage protein, thereby producing a protein antibody conjugate. Although the preparation of the single-chain antibody phage coat protein fusion is described as illustrative, other modalities and fragments thereof are useful as described. In general, an antibody or portion thereof, e.g., a mAb, Fab, or ScFv, is prepared via direct synthesis (e.g., using a PCR extension that overlaps the spliced site, with the additional addition of the restriction sites at the 5 'and 3' ends of the sequence) or they are selected and isolated from the ascites and purified by known methods, such as affinity chromatography. In only the Fab fragment can be used, an antibody cultured from the ascites that is also modified according to normal protocols to produce Fab, eg, through the use of papain digestion. An illustrative antibody that specifically selects the FGF receptor is 11A8 antibody derived from hybridoma 11A8, 11A8 recognizes ECDR1 by Western blot analysis, can immunoprecipitate FGFR from extracts of SK-HEP-1 and SK-MEL-28, a cell melanoma. The 11A8 antibody also stains SK-HEP cells on the cell surface. The receptor binding fragments and variants of 11A8 and the like, the receptor binding antibodies, preferably, antibodies that are also internalized, can also be in non-covalently (covalently) linked to the phage coating in order that it can function as a useful ligand, as described herein. Other antibodies (and fragments thereof) that specifically select the cellular receptors, preferably those antibodies and fragments thereof which bind and internalize, can be identified and synthesized according to the methods described herein using other described methods. in the matter. The amplified light and heavy chain product was cloned into the expression vector. The chosen expression vector preferably contains appropriate promoters and convenient restriction sites. The resulting protein can be expressed optionally as the N-terminal and C-terminal fusion protein. In addition, flexible ligatures can be added between the antibody and the ligand to help favor the appropriate protein. The use of the smaller antibody fragments, e.g., Fab or ScFv, probably facilitates bending still further. The expression, purification and evaluation of the antibody-phage fusion proteins are easily achieved in host cells, eg, in the bacteria, using known protocols. Alternatively, one can be fused with two monospecific antibodies or immunologically active portions thereof with different phage coat proteins, wherein each antibody is directed against the same receptor, or against different receptors. Such constructs may be useful in situations in which the expressed cellular receptors are polymorphic and are used in phage vectors selected from two or more of said polymorphisms that may have a higher probability of delivering the load on the desired cells. The preparation of a fusion between the phage coat protein and the biospecific antibodies are also within the scope of the present invention. As a result, the gene delivery vectors select one or more receptors that can be easily prepared according to the methods described herein. Alternatively, the fused antibody portions are directed to other ligands in opposite manner to specific receptors, instead of targeting antibody / s in the relevant receptors directly. Said methods can be used particularly in situations in which a capacity of ligands to bind to their analog receptor depends on the ability of the ligand to bind a secondary or tertiary structure that could be easily achieved if the ligand is incorporated into a ligand fusion protein. phages For example, chimeric antibodies including receptor binding ligands in the plate of their constant region, such as those described in Published PCT Publication No. WO 91/14438 as described in the published UK Patent Application No. GB 2197323 is also preferred for use as described herein. In addition, bridged antibodies such as those described in the published PCT Application No. WO 92/08801 may be useful in the treatment of conditions in which a "time release" of therapeutic nucleotide sequences is desired. Therefore, the use of bi-specific antibodies that bind to the phage vector to select the cell pending the co-or subsequent administration of vectors carrying highly specific proteases, ribozymes or deoxyribozymes that supply the binding vectors supplying thus, the binding directly into its selected receptor and internalize the vector is also contemplated within the scope of the present invention.

To be useful in the selected bacteriophage vectors, the recombinant fusion protein can bind to the analogous receptor. The fusion proteins can be analyzed for their binding capacity in an ELISA analysis, according to known techniques. To test the functionality of the binding domain receptor, binding and internment of assays that can be carried out on receptor positive cells and binding the specificity were determined by influencing the unlabeled fusion protein as a competitor by normal protocols. In an illustrative method, the internment of a fusion protein is determined by pre-incubating the positive receptor cells with labeled fusion protein by washing the cells to remove the unbound labeled protein, and conditions for further incubations at predetermined temperatures and for various intervals of time. time to allow the internment of the receiver. After the removal of radiolabeled protein bound to the surface, the cells were used and the radioactivity was determined in the cell lysate. The analysis could determine the ability of the fusion protein to bind its analog receptor in the context of a fusion protein. In order to facilitate the binding of a sequence of ligands to a phage sequence, the phage protein can be encoded at the molecular level. Therefore, a nucleotide sequence encoding the therapeutic molecule, toxin, or other regulatory molecule can be operably linked for expression in a phage nucleotide sequence particularly in a sequence encoding the structural protein. For example, protein III or protein VIII of filamentous phages (e.g., M13) can be modified via the binding of a heterologous nucleotide sequence at the C-terminus of the gene encoding the protein. Alternatively, one or more heterologous sequences may be inserted at one site, ie, within the protein sequence with phage coat. Various methods for preparing such fusions are available in the art and are contemplated by the present invention. Finally, it should be appreciated that proteins can be modified via media that are not precisely "immunological" or "genetic". The modification of phage proteins via means other than those exemplified herein are completely within the scope of the present invention. For example, the filamentous phage-based vectors of the present invention may undergo chemical alteration of their coat proteins, e.g., in a way that affects the immunogenicity of the vectors, in order to regulate the absorption and persistence of the vector. in the cells of an individual that are within the therapeutic constructs and compositions were administered. Methods for labeling such alterations for proteins via chemical and physical means (e.g., heat shock) are known to those skilled in the art and can be readily accepted in the relevant literature. '* ~ > »-» * »m-« »" - - ^. '.. 3 «» as. _,. .. j. "M_ m.JatJ .t-JlnS? Tomm.mmm.im, * B. Use of antibodies to ligate the vector and ligands A variation of the procedures described above, which are directed towards the preparation of the fusion proteins, are in the use of bi-specific antibodies or fragments thereof, e.g., in the form of bi-specific ScFv, to select phages in a particular cellular receptor, using an antibody or portion thereof that is raised against the phage-coated protein and the binding of the antibody to one that has been raised against a ligand. Although this is not completely the same as the construction of a fusion protein, several techniques summarized above (e.g., with respect to the preparation of a ScFv) are useful in the preparation of bi-specific antibodies for use as described above. In another embodiment, a fusion protein comprising the phage coat protein and an antibody or mono-specific is useful in ligation of a phage vector for a ligand. The preparation of an antibody or fragment thereof will be readily understood in the art; also see Section A above. The antibody portion of the antibody-fusion protein preferably rises against a preselected ligand, e.g., one of which is not easily incorporated into a fusion protein, perhaps due to conformational difficulties. Such as a fusion with antibody-phage cover can then be used to bind a ligand that selects a specific receptor in the phage vector, for the purposes of delivering a load on a cell expressing the relevant analogue receptor. C. Use of avidin-biotin to bind bacteriophages in the ligand One method to direct phages to cellular receptors is by binding phage to the ligand via avidin-biotin, which can be carried out essentially. A ligand-phage complex is assembled in the presence of the test cells at 0 ° C, and then by incubation at 37 ° C to allow internment. A ligand molecule is conjugated to the biotin in the simple free sulfhydryl group using biotin-BMCC (Pierce, Rockford, IL) according to the protocol suggested by the manufacturer. An unreacted biotin is removed by passing the reaction on the PD-10 desalting column. The cultured cells, e.g., COS cells, were incubated for a predetermined period of time (eg, 24 hours) before the addition of the phages. The cells were washed and biotinylated the ligand was added (often on ice) and the preparation was incubated for a predetermined period of time. Avidin (e.g., Neutravidin, an avidin that spoils the neutral carbohydrate; Pierce, Rockford, IL) was added and the cell was washed repeatedly. The biotinylated anti-phage antibody, such as the anti-M13 antibody, when the phages are M13, was then added and incubated for a predetermined period of time. The washing of the cells was repeated and a sufficient amount of colony forming units was added to reach the receptacle or well, followed by further incubation, preferably on ice. The cells were washed again, resuspended in fresh medium and allowed to incubate further for an appropriate time and at a predetermined temperature. The expression of the agent encoded by the therapeutic nucleic acid sequence carried by the phage vector was then monitored. In several preferred embodiments, the vector further comprises a reporter gene, such as EGFP, which can be easily monitored via fluorescence microscopy or via a fluorescence activated cell sorter (FACS), in accordance with standard techniques. D. Covalent bonds / Chemical conjugation 1. Ligand ligations for bacteriophages using polycations A ligand is linked alternatively to a polycation such as polylysine, which then binds to the phagemid particles as a result of the interaction between the positively charged polycation and the negatively charged phages. In an illustrative procedure, a ligand is covalently linked to polylysine using S-2-pyridyl disulfide (SPDP) according to known protocols (see, for example, Sosnowski et al., J. Biol. Chem. 277: 33647-33653, nineteen ninety six). The phagemid particles are then mixed with ligand-polylysine or polylysine alone and left at room temperature for a predetermined period of time before testing the conjugates in cultured cells, or before for administration ex vivo or in vivo. In order to verify expression, a reporter sequence can be included in the construction of ligand-polylysine-phage; Fluorescent labels such as GFP and fluorescein are particularly useful for in vitro analyzes. In the context of an assay procedure, the treated cells were examined by conventional means, e.g., fluorescent microscopy or FACS. If the reporter sequence is EGFP, self-fluorescent GFP-positive cells were then counted in order to confirm the GFP gene (and nucleotide sequence attached to it) were sequentially translated into cells via the ligated phages for the ligand via polylysine. This method of phage transduction is an attractive alternative for the use of the avidin-biotin system described herein. 2. Ligation of ligands for bacteriophages using interlacing reagents The coat proteins of bacteriophages, preferably of filamentous bacteriophages, can be conjugated directly in a ligand using heterobifunctional crosslinking reagents. For example, a free lysian at the N-terminus of the envelope protein of gene VIII of M13 phages is available from chemical modification (Armstrong et al., EMBO 2: 1641-1646, 1983) and can be conventionally employed for this purpose . In general, the procedure is described as follows.

The phage particles were first thiolated, e.g., via the addition of SPDP, for a predetermined period of time and at a predetermined temperature. An unreacted reagent is removed; the ligand is then reacted with the thiolated phage. The free ligand is removed, and the ligated phages in the ligand are further purified according to the normal protocols. In similar procedures, N-succinimidyl S-acetylthioacetate (SATA) and other suitable heterobifunctional chemical reagents can be used to introduce thiol function, instead of SPDP, in accordance with the recognized methods in the matter. For example, the selected ligation or ligatures are ligated into the ligands bound to the receptor by the chemical reaction, generally reliant on the available thiol or amine group on the ligands bound to the receptors. The ligatures The heterobifunctionals are particularly suitable for chemical conjugation and include such molecules as maleimidobenzoyl-N-hydroxysulfo succinimide ester, N-succinimidyl- (4-iodoacetyl) amino: benzoate, and N-succinimidyl-3- (2-pyridyldithio) propionate. (See, for example, UK Patent Application published Nos. GB 2268492, 2253626 and 2257431, the descriptions of which are incorporated herein by reference). The binding of a ligand to the phage protein is easily confirmed by polyacrylamide gel electrophoresis and immunoassay of phage proteins. For example, under conditions unreduced, the VIII gene protein modified by the ligand was aShn? WÍW- > it changes from its evident molecular mass in an unlinked form to a significantly greater molecular mass when the ligation was successively. The addition to reduce the agent in the sample buffer alters disulfide ligation and yields of free ligands and the protein of gene VIII. The chemically modified ligand-phage constructs can then be analyzed for their ability to translate mammalian cells. Phagemid particles modified by the ligand having an expression cassette containing a promoter and a reporter sequence are added to the plant cells in multiple well plates, for example. The cells were analyzed to mark protein expression at a predetermined interval after the addition of the ligand-phage constructs. 3. Additional ligation methods. Other means of chemically ligating a ligand to a phage particle comprising the expression of a specific reactive portion on the surface of a phage particle, wherein the portion is then specifically and directly conjugated to the selectable ligand. . Examples of reactive portions that are easily treated in the surface protein of a phage particle include several binding proteins, protein A, cysteine and a wide variety of the group of reagents, namely a few examples. (See, for example, published UK Patent Application Nos. GB 2268492 and 2257431, the descriptions of which are incorporated herein by reference.) Methods for treating such portions on the surface of a phage particle are available in the art and have been described in procedural sections. For example, site-specific mutagenesis techniques can be used to alter the amino acid residue sequence of the phage-coated protein, thereby facilitating binding of the ligand at the relevant sites. Next, the preselected ligand (e.g., a polypeptide that binds to an FGF receptor) is conjugated to the phage surface via the reagent portion. In addition to the FGFR binding polypeptides, other useful ligands that can be conjugated to the surface of phage particles include antibodies and fragments thereof (e.g., 11A8), including single chain antibodies, and cysteine, to name a few examples. Still other ligands can bind to the coat proteins on the phage surface. The size of the fusions for coat proteins can be small such as small randomized peptides of 6 (Scott and Smith, Science, 249: 386-390, 1990) for 38 amino acids (Kay, Gen 128: 59-65, 1993) of length of whose peptides that bind to cell surface receptors have been isolated. Protein molecules such as Fab fragments of antibodies (~ 50kDa) have been fused to the phage coat proteins. All proteins such as alkaline phosphatase, trypsin inhibitor of bovine pancreas, trypsin, ß lactamase, cytokine IL3 and glutathione-S-transferase, PDGF receptor (Chiswell and McCafferty, Trends Biotechnol.10: 80, 1992) and IgE ectodomain ( Robertson, J. Biol. Chem. 268: 12736, 1993) have also fused to the surface of phages. For an updated reference list of proteins displayed on phages, see www.unc.edu/depts/biology/bkay/phagedisplay/html. Expression of small peptides on the surface of phage is highly efficient while large peptides are expressed from about 50 copies to less than 1 copy per phage. E. Propagation of Modified Phages Modified phages were propagated as described by Sambrook et al. The suitable host bacterium carrying the F 'episome was developed from an isolated colony to the medium growth phase log. Isolated bacteriophages can be stung by plaques that form on a lawn of development of infected host cells or in semi-solid medium. Turbid plaques developed slowly in infected cells that are visible to the naked eye against a screen of denser infected bacterial colonies. The phage stocks can be prepared in liquid culture from plates isolated from the wells. Approximately 1/10 of the phages from a single plate are used to infect 50 μl of the host bacterium in 2 ml of medium. The culture was incubated at 37 ° C for 5-6 hours with constant agitation. Larger incubation times are avoided to prevent deletion mutants. The bacterium was formed into pellets by microcentrifugation and the supernatant was transferred to a fresh tube. Titration of the 12 phages in the supernatant could be about 10 pfu / ml and can be stored at 4 ° C or indefinitely at -20 ° C. IV. GENE TRANSDUCTION A transduction gene, as used herein, refers to a gene that encodes a detectable product in the target cells. Preferably, the transduction gene is a therapeutic gene. A "therapeutic nucleic acid" or "therapeutic gene" describes any nucleic acid molecule used in the context of the invention that affects a treatment, generally by modifying the transcription or translation of genes. If it includes, but is not limited to, the following types of nucleic acids: nucleic acids that encode, a protein, ribozyme, nonsense nucleic acid, DNA intended to form triple molecules, the protein that binds nucleic acids, and molecules of smaller nucleotides. So, the product of the gene that can be DNA or RNA. These gene sequences can be naturally derived or recombinantly released sequences. A therapeutic nucleic acid can be used to effect gene therapy by serving as a replacement for a defective gene, encoding a therapeutic product, such as TNF, or encoding a cytotoxic molecule, especially an enzyme, such as saporin. Therapeutic nucleic acids can encode all or a portion of a gene and can function by recombining with the DNA around the present in a cell, thus replacing a defective portion of a gene. You can also covet a portion of a protein and . a a * &,. & -, & ? 7- ~ íií * exert its effect by virtue of the co-suppression of a gene product. As described above, the therapeutic gene provides in an operative ligation with a selected promoter, and optionally in operative ligation with other elements involved in transcription, translation, localization, stability and the like. The therapeutic nucleotide composition of the present invention is from about 20 base pairs to about 100,000 base pairs in length. Preferably the nucleic acid molecule is from about 50 base pairs to about 50,000 base pairs in length. More preferably, the nucleic acid molecule is from about 50 base pairs to about 10,000 base pairs in length. Even more preferably, it is a nucleic acid molecule of about 50 pairs at about 4,000 base pairs in length. Nucleic acid compositions can be delivered by the bacteriophages of this invention by a variety of methods including, for example, in vitro, in vivo and ex vivo transduction methodologies. A. Replacement or improvement of genes Nucleic acids for release also include DNA molecules that encode proteins to replace defective genes or provide factors to combat certain diseases or syndromes. Many genetic defects are caused by a mutation in a single gene. The introduction of the wild-type gene can serve to alleviate the deficiency or genetic anomaly. Such genes include HPRT, adenosine deaminase, LDL receptor, factor IX; Factor VIII, growth hormone, von Willebrand factor, PTH (parathyroid hormone), M-CSF, TGF-β, PDGF, VEGF, FGF, IGF, BMP (bone morphogenic protein), VIL type collagen, fibrin, Insulin, behavioral regulator of cystic fibrosis transmembrane, adenosine deaminase and the like. For example, in ischemia, endothelial and smooth muscle cells fail to proliferate. A construct that expresses FGF, alone or in combination with the FGF protein to give short-term release and induce the FGF receptor, can be used to combat the effects of ischemia. In such a case, the FGF gene with a leader sequence to promote secretion that is preferable, well, the FGF gene is preferably driven by a constitutive promoter. In addition, certain angiogenic diseases suffer from an angiogenic factor quality and are therefore deficient in micro-containers. Certain aspects of reproduction, such as ovulation, uterine repair after menstruation and placental development depend on angiogenesis. For reproductive disorders with underlying angiogenic dysfunction, a construct that expresses FGV, VEGF, or other angiogenic factors, may be beneficial. t.i a Cytokine immunotherapy is a modification of immunogenic therapy and involves the administration of tumor cell vaccines that are genetically modified ex vivo or in vivo to express various cytokine genes. In animal tumor models, the cytokine gene transfers the result in significant anti-tumor immune response (Fearon, et al., Cell 60: 387-403, 1990; Wantanabe, et al., Proc. Nat. Acad. Sci. USA, 86: 9456-9460, 1989). Therefore, in the present invention, phages are used to deliver the DNA encoding a cytokine such as IL-12, IL-10, IL-2, GM-CSF, IFN-α. or an MHC gene, such as HLA-B7. The release of these genes can modulate the immune system, increasing the potential for host anti-tumor immunity. Alternatively, DNA that encodes molecules such as ligands B7.1 and B7.2 costly for CD28 and CTLA-4, respectively, can also be delivered to improve the immunity measured by T cells. These genes can be co-delivered with cytokine, using the same or different promoters and optionally with an internal ribosome binding site. Similarly, the expression of a-1, 3-galactosyl transferase on tumor cells allows cell death measured by complement. Thus, acquired or complex multi-specific diseases, such as erythropoietin deficiency induced by renal failure, Parkinson's disease (dopamine deficiency), adrenal insufficiency, immune deficiencies, cyclical neutropin, could be treated using a therapeutic gene released by a ligand In some cases, vascular growth is convenient. Since smooth muscle cells are below the vasculature, release of endothelial growth factors, such as FGF, especially FGF-2, VEGF, junction 1, and junction 2, through smooth muscle cells are advantageous. B. Genes that covet protein cytokines (including prodrugs) An agent that covets the cytocide is a nucleic acid molecule (e.g., DNA or RNA) that, up to internment by a cell, and subsequent transcription (it is DNA) and / or translation in a product that is cytotoxic or cytostatic to a cell, for example, by inhibiting cell growth through interference with protein synthesis or through disruption of the cell cycle. Said product can activate by dividing the rRNA or ribonucleoprotein, by inhibiting an elongation factor, by dividing the rRNA, or by other mechanisms that reduce the synthesis of protein at a level such as the cell that does not survive. The product can be a protein, ribozyme, deoxyribozyme, counter-sense and the like. Examples of suitable products include, without limitation saporin, resins, abrin, or other ribosome inactivation proteins (RIPs), Pseudomonas exotoxin, DNA inhibitors, RNA or protein synthesis, nonsense nucleic acids, other metabolic inhibitors ( v., DNA dividing the molecules), prodrugs (e.g., HSV thymidine kinase and cytosine deaminase bacteria), light activated porphyrin, ricin, ricin A chain, maize RIP, felonin, diphtheria toxin , chain of diphtheria toxin A, trichosantin, tritin, antiviral protein of grana (PAP), antiviral protein mirabilis (MAP), Diantinis 32 and 30, abrin, monordin, bryodin, shiga, a catalytic anhydride of seed protein biosynthesis of cucumber (see, for example, WO 93/24620), Pseudomonas exotoxin, biologically active fragments of cytokines and others known to those skilled in the art. Preferred were DNA molecules that encode an enzyme that result in cell death or that return the cell susceptible to cell death by the addition of another product. Ribosome inactivation proteins (RIPs), which include ricin, abrin, saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes, proteins that inactivate the ribosome are inactive ribosomes that interfere with the protein extension step of synthesis of protein. For example, the ribosome saporin inactivation protein is an enzyme that divides rRNA and inhibits protein synthesis. Other enzymes that inhibit protein synthesis are especially suitable for use in the present invention. Any of these proteins, if not derived from mammalian sources, can use mammalian preferred codons. The preferred codon used is exemplified in Current Protocols in Molecular Biology, infra, and Zhang et al. (Gen 705: 61, 1991).

A nucleic acid molecule encoding a prodrug can alternatively be used within the context of the present invention. The prodrugs are inactive in the host cell until a substrate or an activation molecule is provided. As used herein, a "prodrug" is a compound that metabolizes or in some manner converts an inactive, non-toxic compound to a biological, pharmaceutical and therapeutic form of the active toxic compound or is modified upon administration to a compound active through metabolic or other processes. More typically, a prodrug activates a compound with little or no cytotoxicity in a toxic compound. Two of the most commonly used prodrug molecules, both of which are suitable for use in the present invention, are HSV thymidine kinase and E. coli cytosine deaminase. In summary, a wide variety of gene products that directly or indirectly activate a compound with little or no cytotoxicity in a toxic product can be used within the context of the present invention. Representative examples of such gene products include HSVTK (herpes simplex virus thymidine kinase) and VZVTK (varicella zoster virus thymidine kinase), certain purine arabinosides are selectively phosphorylated and substituted for pyrimidine compounds. Phosphorylation converts these compounds to metabolites that are cytotoxic or cytostatic. For example, the exposure of the drugs ganciclovir, acyclovir, or any of their analogues (e.g., FIAU, FIAC, DHPG) for cells expressing HSVTK allows the conversion of the drug into its corresponding active nucleotide triphosphate form. Other gene products that can be used within the context of the present invention include guanine phospho-silsyl transferase from E. coli, which concerns thioxanthin in toxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol. 7: 4139-4141, 1987 ); alkaline phosphatase, which concerns inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin phosphate to toxic dephosphorylated compounds; cytosine deaminase fungal (eg, Fusarium oxysporum) or bacterial, which concerns 5-fluorocytosine of the toxic compounds of 5-fluorouracyl (Mullen, PNAS 89:33, 1992), carboxypeptidase G2, which divides glutamic acid from glutamic acid of para-N-bis (2-chloroethyl) aminobenzoyl, creating toxic benzoic acid mustard; and Penicillin-V amides, which convert phenoxyacetamide derivatives of doxorubicin and melphalan to toxic compounds (see generally, Vrudhula et al., J. of Med. Chem. 36 (7): 919-923, 1993; Kern et al. Immun Immunother, 37 (4): 202-205, 1990). In addition, a wide variety of Herpesviridae thymidine kinases, including both primate and non-primate herpesviruses, are suitable. Such herpesviruses include Herpes Simplex Virus Type 1 (McKnight et al., Nuc Acids, Res 8: 5949-5963, 1980), Herpes Simplex Virus (Swain and Galloway, J. Gen. Virol 46: 1045-1050, 1983). , Varicella Zoster Virus (Davison and Scott, J. Gen. Virol. 67: 1759-1816, 1986); herpesvirus of marmosets (Otsuka and Kit, Virology 135: 316-330, 1984), feline herpesvirus type 1 (Nunberg et al., J. Virol. 63: 3240-3249, 1989), pseuorabies virus (Kit and Kit, U.S. Patent No. 4,515,497, 1985), choline herpesvirus type 1 (Robertson and Whalley, Nuc.Aids Res. 76: 11303-11317, 1988), bovine hepesvirus type 1 (Mittal and Field, J. Virol 70: 2901 -2918, 1989), turkey herpesvirus (Martin et al., J. Virol. 63: 2847-2852, 1989); Marek's disease virus (Scott et al., J. Gen. Virol. 70: 3055-3065, 1989), saimiri herpesvirus (Hones et al., J. Gen. Virol. 70: 3003-3013, 1989) and Epstein-Barr (Baer et al., Nature (London) 370: 207-311, 1984). Such herpesviruses can be easily obtained from commercial sources such as the American Type Culture Collection ("ATCC"; Manassas, GOES). In addition, as indicated above, a wide variety of inactive precursors can be converted into active inhibitors. For example, thymidine kinase can be phosphorylated nucleosides (e.g., dT) and nucleoside analogs such as ganciclovir (9- { [2-hydroxy-1- (hydroxymethyl) ethoxymethyl) guanosine), famciclovir, bucyclovir, penciclovir, valciclovir, acyclovir (o- [2-hydroxy ethoxy) methyl] guanosine), tri-fluoro-thymidine, 1- [2-deoxy-2-fluoro-beta-Da rabino furanosyl] -5-yodouracil, ara-A (adenosine arabinoside, vivarabine), 1-beta-d-arabinofuranoxil thymine, 5-ethyl-2'-desoxirubicin, 5-iodo-5'-amino-2,5'-dideoxirubicin, idoxuridine (5-iodo-2'- deoxyuridine), AZT (3'-axido-3 'thymidine), ddC (dideoxycytidine), AlU (5-iodo-5' amino 2 ', 5'-dideoxyuridine) and AraC (cytidine arabinoside). Other gene products can return to a cell susceptible to toxic agents. Such products include tumor necrosis factor, viral proteins, and channel proteins that transport drugs. In addition, a cytocidal coding agent can be constructed as a pro-drug, which when expressed in the appropriate cell of the type that is processed or modified for an active form. For example, the saporin gene can be constructed with an extension of the N or C terminus containing the protease sensitive site. The The extension makes the initially transferred inactive protein and subsequent division in a cell that expresses the appropriate protease that restores the enzymatic activity. The DNA sequences of these products are well known (see Bank of Genes). A nucleic acid molecule that encodes of which can be isolated by normal methods such as amplification (e.g., PCR), probe hybridization of genomic or cDNA libraries, antibody protections of expression banks, chemically synthesized or obtained from commercial or different sources. The additional types of cytocides that can be delivered according to the methods of the present invention are antibody molecules that are expressly expressed within the target cell; therefore, these antibody molecules have been known as "intrabodies". Conventional methods of preparation antibodies and sequencing are useful in the preparation of intrabodies and nucleic acid sequences encoding the same; if it is the site of action of the intrabodies that confer particular novelty on said molecules. (For a review of various methods and compositions useful in the modulation of protein function in cells via the use of intrabodies, see published International Application No. WO 96/07321). Intrabodies are antibodies and antibody derivatives (including single-chain antibodies or "SCA") introduced into cells as transgenes that bind to and incapacitate an intracellular protein in the cells expressing the antibodies. As used herein, the intrabodies encompass monoclonal single chain antibodies, V regions and the like, throughout the binding to the target protein. Intrabodies for proteins involve cell replication, tumorigenesis and the like (e.g., HER2 / neu, VEGF, VEGF receptor, FGF receptor, FGF) are especially useful. For example, antibodies to HER2 / neu (also called HER2) can be used to inhibit the function of this protein. HER2 / neu has a pivotal role in the progression of certain tumors, human breast carcinoma, non-small ovaries and lung. Therefore inhibiting the function of HER2 / neu may result in slow or arrested tumor development (e.g., U.S. Patent No. 5,587,458). In view of the fact that HER2 / neu is a receptor protein, and can function as the "target" of a ^^^^^^^^^ * ^. conjugated antibody on the surface of a phage particle, as described further in this application. C. Contrasent and ribozymes The conjugates provided herein may also be used to deliver a ribozyme, counter-sense and the like for the selected cells. Such products include nonsense RNA, nonsense DNA, ribozymes, deoxyribozymes, triple-forming oligonucleotides, and oligonucleotides that bind proteins. Nucleic acids can include RNA trafficking signals, such as viral packaging sequences (see, for example, Sullenger and others Science 262: 1566, 1949). Nucleic acids and oligonucleotides for use as described herein may be synthesized by any method known to those skilled in the art (see, for example, WO 93/01286, US Application Serial No. 07 / 723,454; 5,218,088; 5,175,269; 5,109,124). The identification of the oligonucleotides or ribozymes to be used as counter-sense agents and the DNA encoding the genes for selecting the gene therapy release involve methods well known in the art. For example, the convenient properties, lengths and other characteristics of said oligonucleotides are well known. The antisense oligonucleotides can be designed to resist degradation by the endogenous nucleolytic enzymes using ligations such as phosphorothioate, methylphosphonate, sulfones, sulfate, cetyl, phosphorodithioate, phosphoramidate, phosphate esters and the like (see, for example, Stein in: Oligodeoxinucleotides.) Antisense Inhibitors of Gen Expression, Cohen, De, Macmillan, Press, London, pp. 97-117, 1989); Jager et al., Biochemistry 27: 7237, 1988). Counter-sense nucleotides are oligonucleotides that bind in a form specified for sequence in the nucleic acids, such as mRNA or DNA. When they bind to the mRNA that has complementary sequences, the translation that avoids the contradiction of the mRNA (see, for example, U.S. Patent Nos. 5,168,053, 5,190,931, 5,135,917, 5,087,617). Triple molecules refer to DNA strands that bind to duplex DNA to form a triple colinear molecule, thus preventing transcription (see, for example, U.S. Patent No. 5,176, 996).

Particularly useful counter-sense nucleotides and triple molecules are molecules that are complementary or bind to the sense strand of DNA or mRNA that encode a protein involved in cell proliferation, such as an oncogene or growth factor, (e.g. bFGF, int-2, hst-1 / K-FGF, FGF-5, hst-2 / FGF-6, FGF-8). Other useful antisense oligonucleotides include those specific for IL-8 (see, e.g., U.S. Patent No. 5,241,049), c-src, c-fos H-ras (lung cancer), K-ras (breast cancer) , urokinase (melanoma); BCL2 (T-cell lymphoma), IGF-1 (glioblastoma), IGF-1 receptor (glioblastoma), TGF-β, and CRIPTO EGF receptor (colon cancer). These contradictory plasmids reduce tumorigenicity in nude and syngeneic mice.

A ribozyme is an RNA molecule that specifically divides RNA substrates, such as mRNA, that result from inhibition or interference with cell development or expression. There are at least five known classes of ribozymes that involve the cleavage and / or ligation of RNA strands. The ribozymes can select any RNA transcript and can catalytically divide the transcript (see, for example, US Patent No. 5,272,262, US Patent No. 5,144,019, and US Patent Nos. 5,168,053, 5,180,818, 5,115,742 and 5,093,246). . In addition, inhibitors of inducible nitric oxide synthase (NOS) and endothelial nitric oxide synthase are cytokines which are useful for delivering the cells. Nitric oxide (NO) is involved to be involved in the regulation of vascular development and tones in arteriosclerosis. NO is formed from L-arginine by nitric oxide synthase (NOS) and modulated immune, inflammatory and cardiovascular responses. As described herein, the phage vectors of the present invention are used to deliver a variety of therapeutic sequences for target cells. In various embodiments, the therapeutic sequences encoded or comprising the enzymatic DNA and / or the RNA molecules that are capable of dividing other nucleic acid molecules, including the DNA molecules, RNA molecules and hybrids thereof. Therefore, expression vectors useful in such embodiments are also contemplated therein (see, for example, International Published Application Nos. WO 92/06693, WO 96/17086, and WO 95/31551; US Nos. 4,987,071, 5,580,967 and 5,595,873, the descriptions of which are incorporated by reference herein). Briefly, using the enzymatic RNA molecules ("ribozymes") as an example, a method of forming an expression vector of the enzymatic RNA molecule includes providing a vector comprising the nucleotide acid encoding a first ribozyme and providing a molecule of single-stranded DNA encoding a second ribozyme. The single-stranded DNA then leaves for quenching to form the partial double DNA that can be filled by treatment with an appropriate enzyme, such as a DNA polymerase in the presence of dNTP, to form a duplex DNA that can be ligated into the vector . Larger vectors resulting from the use of this method can be selected to ensure a large number of copies of the single-stranded DNA encoding the second molecular enzyme RNA that is incorporated into the vector. Suitable restriction endonuclease sites are also provided to facilitate the construction of the vector in the DNA vectors or in the DNA vectors required by an RNA expression system. The present invention also relates to expression vectors that include a nucleic acid segment in the molecular DNA or RNA, preferably in a form that allows the expression of the DNA molecule within a target cell. Therefore, in general, a useful expression vector together with ribozymes or deoxyribozymes that include a bacterium, viral or eucharistic promoter within a plasmid, cosmid, phagemid, virus, viroid, or phage vector. Other suitable vectors include double stranded DNA (dsDNA) partial double stranded DNA, dsRNA, partially dsRNA, or single stranded RNA (ssRNA) or DNA (ssDNA). It could also be appreciated that they are useful vectors according to the present invention that are not necessarily circular. It is also preferred that a nucleotide sequence encoding the enzymatic NA molecule is transcriptionally ligated into a promoter sequence. For example, a vector according to the present invention comprising an enzyme RNA molecule under the control of a viral promoter, such as the Epstein-Barr virus (EBV) promoter. A variety of viral promoters useful for this purpose are well known in the art, see, for example, those described in published PCT Application No. WO 93/23569, the descriptions of which were incorporated by reference herein. In another variation, one or more nucleotide sequences that encode the additional enzymatic RNA molecule are also included in the vector; the sequences that covet the additional enzymatic RNA molecule can be located on the side, or both sides, of a nucleotide sequence encoding the first enzyme RNA molecule. Preferably, intervening nucleotides or nucleotide sequences exist between the sequences encoding the successive enzymatic RNA molecule. If the release of a vector comprising a ribozyme or deoxyribozyme construct for a eukaryotic cell is desired, the cellular splicing mechanisms within the target cells can be used or integrated to divide into the enzymatic RNA molecules encoding the recognition sequences for the second molecular enzyme RNA within the flanking sequences of the expressed transcript. Multiple copies of the release of the first enzyme RNA molecule can be provided to improve the release of the second enzymatic (ie therapeutic) RNA molecule if the billing rate is slower than the degradation rate of the second RNA molecule enzymatic A method of forming the expression vectors of the enzyme RNA molecule and for the production of enzymatic RNA molecules are further described in the published publications mentioned above and patents filed, the descriptions of which are incorporated by reference herein. . V. FORMULATIONS AND ADMINISTRATION OF RELEASE VEHICLES The conjugates and complexes provided herein are useful in the treatment and prevention of various diseases, syndromes and hyperproliferative disorders, such as restenosis, or other smooth muscle cell diseases, tumors, such as melanomas. , ovarian cancers, neruoblastomas, pterygium, secondary lens nebulosity and the like. As used herein, "treatment" means any form in which the symptoms of a condition, disorder or disease are improved, or in some way, altered beneficially. The treatment also encompasses any pharmaceutical use of the compositions herein. As used herein, "decrease" of the symptoms of a particular disorder refers to any decrease, which are permanent or temporary, duration or compromises, which can be attributed or associated with the administration of the composition. In certain embodiments, the compositions of the present invention can be used to treat angiogenesis-dependent diseases. In these diseases, vascular development is excessive to allow the desired development of other tissues providing blood supply. These diseases include angiofribroma, arteriovenous, malformations, arthritis, atherosclerotic plaques, corneal graft neovascularization, delayed wound healing, diabetic retinopathy, granulations due to burns, hemangiomas, hemophilic junctions, hypertrophic scars, neovascular glaucoma, fractures without union, Osler-weber syndrome, psoriasis, pyogenic granuloma, retrolental fibroplasia, scleroderma, solid tumors, trachoma, and vascular adhesions. sS ^ ltéaBJ ^ g ^ g ^ M ^ SMa ^ g ^^^^^^^^ Inhibiting vessel formation (angiogenesis), the desired development can be slow or stopped, thus improving the disease. In a normal vessel, a single layer of lumen endothelial cell lines and the development of the receptor requires the proliferation of endothelial cells and smooth muscle cells. Also, the phage of the present invention can be used to treat tumors. In these diseases, cell growth is excessive and uncontrolled. Suitable tumors for treatment within the context of this invention include, but are not limited to, breast tumors, gliomas, melanomas, prostate cancer, hepatomas, sarcomas, lymphomas, leukemias, ovarian tumors, thymomas, nephromas, pancreatic cancer , colon cancer, head and neck cancer, stomach cancer, lung cancer, mesothelioma, myeloma, neuroblastoma, retinoblastoma, cervical cancer, uterine cancer, and squamous cell carcinoma of the skin. For such treatments, ligands are chosen to bind cell surface receptors that are generally expressed in tumors. By supplying the compositions of the present invention, the unwanted growth of cells can be slowed down or stopped, thus improving the disease. The methods used herein are specifically directed to, and kill or stop the proliferation of tumor cells that have the receptors for the ligand on their surface.

Phages are also used to treat or prevent arteriosclerosis and stenosis, a process and the resulting condition that occurs after angioplasty in which the arteries become clogged again. Generally, atherosclerosis treatment involves the enlargement of a supporting vascular lumen, allowing for greater blood flow and oxygenation for the distal tissue. Unfortunately, these procedures induce normal wound healing in response in the vasculature resulting in restenosis. Of the three components for the normal vascular response to damage, thrombosis, elastic rebound and proliferation of smooth muscle cells, antithrombotic / platelet inhibitors and vascular supports, they are effectively directed to acute / sub-acute thrombosis and elastic rebound, respectively . However, therapy can not modify vascular remodeling, that is, due to the proliferation of smooth muscle cells in the lesion, its deposition of extracellular matrix and the subsequent formation of a neointima. Consequently, the phage could be used to deliver the therapeutic nucleic acids or proteins that can inhibit restenosis. Wound response also occurs after other interventions, such as coronary balloon angioplasty and peripheral receptors, with or without support; carotid endarterectomies; vein grafts; and synthetic grafts in peripheral arteries and arteriovenous shunt. Although the course of time of response to injuries is not well definedIf the answer can be suppressed in a short time (approximately 2 weeks), a long-term benefit is achieved. In certain embodiments, the bacteriophages of the present invention can be used to treat a variety of genetic abnormalities, such as deficiencies of important functional enzymes. Examples of such diseases include Gaucher's disease (glucocerebrosidase deficiency), mucopolysaccharidosis (beta-glucuronidase deficiency), hyperammonemia (deficiency of orinithine transcarbamylase) and others. One skilled in the art will appreciate that the phage of the present invention can be delivered in vivo by administration to an individual, usually via systemic administration (e.g., oral, intravenous, interaperitoneal, intramuscular, subdermal, or intracranial fusion) or topical application (e.g., membranes of the dermis or mucous membranes). Using the selected ligands, the bacteriophages of the present invention are capable of selecting a specific cell type for transduction. Alternatively, the phage of the present invention can be delivered to cells ex vivo. The ex vivo treatment provides for the removal or removal of a population of cells from a patient, transduction with bacteriophages containing therapeutic nucleic acids, and re-implantation in a patient's body. Illustrative procedures for carrying out generalized ex vivo gene therapy as described in US Pat. Nos. 5,399,346 and 5,665,350. In addition, cells that have been translated with phage ex vivo can be re-introduced into the patient or animal as described in a variety of methodologies. See, for example, the Patents of E.U.A. Nos. 5,399,346, 5,665,350, PCT Publication NO. WO 92/14676 (claim priority for U.S. Patent No. 667,169), PCT Publication No. WO 90/06997 (priority claimed in U.S. Patent No. Serial No. 283,586). Exemplary cell populations, for ex vivo transduction, include bone marrow cells, liver cells, pancreatic cells, hematopoietic cells, and umbilical blood and cells. Said cells can be processed to purify the desired cell population for transduction, for example to obtain the specific subgroup of the transducing cells. Preferred methods of purification include various cell sorting techniques, such as antibody screening, FACS and affinity chromatography using the coupled matrix for antibodies specifically reactive with the desired subgroup of cells. The isolated cells can be translated and re-introduced to a patient or, alternatively, the cells can be expanded in the culture before re-introduction. A. Preparation of pharmaceutical agents & Pharmaceutical carriers or vehicles suitable for the administration of the conjugates and complexes provided herein include any of the carriers known to those skilled in the art which are suitable for the particular mode of administration. In addition, bacteriophages can be formulated as the plant of the pharmaceutically active ingredient in the composition or can be combined with other active ingredients. The bacteriophages can be administered by any appropriate route, for example, orally, parenterally, including intravenously, intradermally, subcutaneously or topically, in liquid, semi-liquid or solid form and are formed in a suitable form for each administration route. The preferred modes of administration depend on the indication treated. The bacteriophages can be formulated in the pharmaceutical compositions suitable for local, intravenous and systemic application. Release formulations of the type are also suitable. The effective concentrations of the bacteriophages are mixed with a suitable carrier or pharmaceutical carrier. As used herein an "effective amount" of a compound to tare a particular disease is an amount that is sufficient to improve, or in some ways reduce the symptoms associated with the disease. Said amount can be administered in a single dose or can be administered according to the regimen, so it is effective. The quantity can cure the disease but, normally, it is administered in order to improve the symptoms of the disease. Repeated administration may be required to achieve the desired improvement of symptoms. The phage vectors of the present invention are particularly well suited for oral administration and have the ability to deliver the sequences of therapeutic genes on the systemic level, not similar to the gene delivery vectors currently tested and in use. As previously observed, phage is able to withstand strong environments and conditions such as those common to the digestive tract of mammals; therefore, they are ideally suited for oral / systemic formulations and administration. An additional advantage of the currently described vectors is the fact that many of the ligands are described as preferred embodiments of the present are resistant to proteases; said ligands are particularly preferred for use in formulations and compositions designed for oral / systemic administration. Therapeutically effective concentrations and amount can be determined empirically by testing the conjugates and complexes in known in vitro and in vivo systems, such as those described herein; Doses for humans or other animals that can be extrapolated from them. The bacteriophages are included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the treated patient. The phages can be supplied as pharmaceutically acceptable salts, esters or other derivatives of the conjugates including any salt, esters or derivatives that can be readily prepared by those skilled in the art using known methods for such derivation and which produce the compounds that can be administered animals or humans without substantial toxic effects. It should be understood that the number and degree of side effects depend on the condition for the conjugates and complexes to be administered. For example, certain toxic and undesirable side effects are tolerated when treating life-threatening diseases, such as tumors, which can not be tolerated when treating laser-related disorders. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include any of the following components: a sterile diluent, such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA), regulatory solutions, such as acetate, citrates and phosphates; and agents for adjusting toxicity such as dextrose or chloride. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass, plastic or other suitable material.

If administered intravenously, suitable carriers include physiological saline or phosphate buffer saline (PBS) and solutions containing thickeners and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof. the same. Liposomal suspensions are also suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. Phages can also be mixed with other active materials that do not affect the desired action or with materials that supplement the desired action. The phage can be prepared with vehicles that protect against rapid elimination from the body, such as type-release formulations or coatings. Such vehicles include controlled release formulations, such as, but not limited to, microencapsulated implant and release systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acids, polyorthoesters, polylactic acids and others. For example, the composition can be applied during surgery using a sponge, such as a commercially available surgical sponge (see, for example, Patents of E.U.A. Nos. 3,956,044 and 4,045,238) that have been soaked in the phages. Phages can also be applied in pellets (such as Elvax pellets of the ethylene vinyl acetate copolymer resin). Preferred vehicles in this regard include gene-activated matrix (GAM), as described in PCT Publication No. WO 97/38729 (priority claimed in US Patent No. 08 / 199,780 and 08 / 316,650). In summary, an activated gene matrix is any matrix material that contains the DNA encoding a therapeutic agent, for example, bacteriophages of this invention. In addition, said gene-activated matrices, preferably, are derived from any biocompatible material. If the oral administration is desired, phages could be provided in a composition that projects from the acidic environment of the stomach, for example in the enteric coating or in combination with an antacid. Alternatively, freeze dried bacteria that is "infected" with phages that can be used for oral administration purposes, which have the ability to spread in the intestine and supply the bundle. Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. Tablets, pills, capsules, troches and the like containing any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, stearate magnesium; a slider such as, but not limited to, dioxide > «? itft "-iHfai" * - > «> * T.i ?? r., a-A S. colloidal silicone; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate and fruit flavoring. When the dose unit is a capsule, it may also contain the material of the above type, a liquid vehicle such as fatty acid. Finally, the compounds can be packaged as articles of manufacture containing packaging material, one or more conjugates and complexes or compositions as provided herein within the packaging material and a label indicating the indication for which the conjugate is provided, H.H. Testing constructs Nucleic acid release vehicles can be evaluated in any number of in vitro and in vivo model systems. As described in the patent and shown in the examples, phages could bind in a cell in a specific way and internalize (similarly through the endosomal pathway). In addition, the transgene should be expressed by the host cells (selected). Therefore, appropriate analysis includes, without limitation, a binding assay to verify that the phages bind to the target cells, a displacement analysis to show that the phages can be spliced by excess free ligand, an internment analysis that show that phages enter (Barry et al., supra); Dunn, supra; and Hart et al., supra) and a functional analysis to show that the transgene is activated in the host cell (see, for example, US Patent Series No. 09 / corresponding to US Provisional Series No. 60 / 057,067) . The following examples are offered by the form of illustration, and not by the form of limitation. EXAMPLES EXAMPLE 1 PREPARATION OF PHAGEMID PARTICLES The phagemid pEGFP-N1 (Clontech; Palo Alto, CA) containing a green fluorescent protein (GFP) gene under the control of the readily immediate CMV promoter. The CMBV promoter is highly active in a wide variety of mammalian cell lines, in addition, other promoters of mammalian cells are used. The phagemid RC-CMVb is a derivative of pRC-CMV (InVitrogen, San Diego, CA). The gene lacZ £. coli was inserted into the pGR-CMV phagemid at the EcoRV site of the low-current multiclonation site of the CMV promoter to form pRC-CMVb. Phagemids containing three origins of DNA replication: the origin of pUC for replication as a double-stranded plasmid in E. coli; the origin f1 for replication as the DNA of single-stranded phage, and the origin of SV40 for high-copy replication in COS-1 (containing the large T antigen) of mammalian cells. They also contain the neomycin phosphotransferase gene, regulated by the early SV40 gene, which confers resistance to the antibiotic G418 when it is expressed in > ....... ..¿ "....... (tota,». mammals (Southern and Berg, J. Mol, Appl. Genet, 7: 327-341, 1982). The phagemid pEGFP-N1 is propagated in the host strain of E. coli, DH5aF 'to leave for higher infection with the auxiliary bacteriophages M13, M13K07. Phagemid particles are prepared according to known methods (see, for example, Sambrook et al., Molecular Cloning: A Laboratoy Manual, Cold Spring Harbor Press, 1989, and Rider et al., In Phage Display of Peptides and Proteins: A Laboratory Manual., Academic Press, 1996). A colony of fresh bacteria containing pEGFP-N1 are suspended in 3 ml of 2X YT medium, M13K07 was added at the end of the titre of 2 X 107 pfu / ml and the culture was incubated for 1 hour at 37 ° C. Kanamycin was added at the end of the 70 μg / ml concentration and incubation continued for 14-18 hours. The bacterial culture was pelleted at 12,000 x g for 10 minutes, and the supernatant was transferred into a fresh tube. The phagemid particles are precipitated by the addition of 1/3 volume of 30% polyethylene glycol (PEG) -1.5M NaCl. The solution was stirred and left for 1 hour once in yarn or overnight at 4 ° C. The phages were pelleted by centrifugation at 12,000 x g for 10 minutes at 4 ° C. The pellet drained strongly and resuspended in 1/10 original volumes of PBS (phosphate buffer pH 7.4). The precipitation of PEG was repeated and the pellet resuspended in 1/20 volumes of PBS. The phagemid suspension was pasteurized by heat at 65 ° C for 5 minutes, filtered through a 0.45uM filter, and stored at 4 ° C. EXAMPLE 2 PREPARATION OF MODIFIED VECTORS FOR MAMMAL CELL TRANSDUCTION A mammalian expression cassette was inserted into a phage or phagemid and used to detect the ligand mediated by the input phages via expression of the reporter gene in mammalian cells. A vector of type 3 filamentous phage was modified for transduction of mammalian cells by insertion of a GFP expression ribbon consisting of a CMV mammalian transcriptional promoter, the green fluorescent protein gene of pEGFP-N1 (Clontech , Palo Alto, CA), and a transcriptional terminator of the bovine development hormone and the polyadenylation signal to mark the vector, MEGFP3 (see Figure 1A). The mammalian expression cassette also contains an SV40 origin of replication adjacent to the CMV promoter. Similar constitutions for monitoring the entry and subsequent expression of phage genomes in mammalian cells are made up of other known phage or phagemid vectors including pCANTB 5 E (Pharmacia Biotech, Piscataway, NJ) or M13 type 3 or 33 for lll gene fusions (see Kay, BK and other Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, 1996, McConnell, SJ et al., Mol. Divers. 7: 165-176, 1996) and the M13 vector of type 8 or 88 for fusions for the gene VIII protein (Roberts et al., Methods Enzymol, 267: 68-82, 1996; Markland, W. et al., Gen 709: 13-19, 1991). EXAMPLE 3 FGF LIGATURE FOR FAGEMIDE PARTICLES USING Avidin-Blotin A method for selecting phages from FGF receptors is linked to the phage for FGF via avidin-biotin. This complex of FGF bundle was assembled in the presence of test cells at 0 ° C, and then by incubation at 37 ° C to allow internment. A mutated FGF, FGF2-3 (Cys in residue 96 is mutated in Ser) (Sosnowski et al., J. Biol. Chem. 277: 33647-33653, 1996) are conjugated to biotin in the free simple sulfhydryl group using biotin -BMCC (Pierce; Rockford, IL) according to the protocol suggested by the manufacturer. The unreacted biotin is removed by passing the reaction on the PD-10 desalting column. While relevant, biotinylated transferrin is available from Sigma (St. Louis, MO) and biotinylated EGF is sold by Molecular Probes (Eugene, OR). COS cells were incubated in 12-well plates at 20,000 cells / well for approximately 24 hours before the addition of phages. Cells were washed twice in PBS / FBS cooled on ice (PBS with 2% fetal bovine serum). After 1.2 μg of B-FGG (biotinylated FGF) they were added on ice for 15 minutes. Neutravidin (NAV; avidin which disposes of the carbohydrate; Pierce, Rockford, IL) was added at 10 μg / ml in 1 ml of PBS / FBS for 15 minutes in ice. The washed cells were repeated and biotinylated anti-M13 antibody (sheep anti-M13-biotin, Prima 5 Prima, Inc., Boulder, CO) was added at 10 μg / ml in 1 ml of PBS / FBS for 15 minutes. minutes on ice. The washed cell was repeated and the colony forming units 108 were added for each well in 1 ml of PBS / FBS for 1 hour in yarn. The cells were washed again, resuspended in fresh medium and returned to the incubator at 37 ° C. The expression of GFP in cells was monitored by fluorescence microscopy (Nikon Diaphot inverted microscopy) using the fluorescein filter set (FITC). In one experiment, approximately 30-35 cells expressing GFP from autofluorescence cells were observed per well after 72 hours. A few autofluorescent cells were observed as the number of phages was decreased. Non-fluorescent cells were positively observed at 105 CFU or less phage per well. The results of control experiments are shown in Table 1. Treatment with the single-stranded nuclease (seed nuclease, New England Biolabs, MA) does not significantly affect the efficiency of GFP expression indicating that the transfection occurs via the factor intact and was not due to the free contamination of single-stranded phage genome DNA. Similar results are obtained using Dnasa I, a nuclease of a single strand and double strand, instead of between the nuclease seed. The addition of 2 μg of EGFP-N1 DNA instead of the phage do not result in any of the GFP-positive cells, which indicate the GFP infection and is not due to the internment of the plasmid contaminating the DNA in the preparations of the phages Table 1 As indicated, the appearance of cells expressing GFP are dependent on the presence of biotinylated FGF, NAV and anti-M13 antibody (Table 1). Including biotinylated FGF and NAV alone or anti-M13 and NAV only result in greatly reduced transfection efficiency (1 positive cell versus 35 per well). Non-positive cells are observed when the NAV addition step is omitted or when equivalent titers of phages alone are added. When free (non-biotinylated) FGF is added to compete with the biotinylated FGF, phage absorption is inhibited (Figure 2). Free FGF was added for 4 minutes before biotinylated FGF at increased concentrations. As presented in Figure 2, the number of autofluorescent cells and inhibit in a dependent manner so that more FGF is added. These results indicate that the internment of the phages is mediated by the interaction of the biotinylated FGF with its receptor. In another experiment, COS cells were incubated in 6-well plates at densities of 40,000 cells / well for 24 hours before addition phages. The phage complex FGF2 was assembled on the cell surface, using a modification of the recognition described by Moro et al., For the tumor the selected antibody (Moro et al., Cancer Res. 47: 1922-128, 1997). Cells were washed twice with ice-cold PBS / FBS (PBS with 2% fetal bovine serum) and biotinylated FGF, biotinylated transferin, or biotinylated EGF were added for 15 minutes. The avidin scored with neutral carbohydrate (Pierce, Rockford, IL) was then added with the binding of biotinylated FGF2, on the cell surface in PBS / FBS cooled on ice for 15 minutes and the cells were washed again. Fifteen minutes later an aliquot of biotinylated anti-M13 antibody (anti-M13-biotin from Rabbit, Sigma, St. Louis, MO) (30 μg / ml) was added to react with the FGF2 complex biotinylated with NAV on the cell surface, the cells were washed and the phages were added in PBS / FBS. After one hour on ice, the cells were washed, in fresh culture medium and returned to the incubator at 37 ° C to allow internment. In some cases chloroquine (50-100 μM) was included in the medium for 2 hours after phage removal. Both GFP and β-galactosidase expression were analyzed in the phagemid of the treated particle and the COS-1 control cells at 72 hours after the addition of the phagemid particles. The expression of GFP in phage-treated cells was measured by counting the number of autofluorescent cells. Cells were washed twice in PBS / FBS and 1 ml of Dulbecco's PBS was added to each well. Fluorescent cells were detected directly on the culture plates using an inverted epifluorine microscopy (Nikon Diaphot) equipped with fluorescence isothiocyanate filter set (FITC). The GFP experiments were done in triplicate and repeated at least once. The β-galactoside in the lysates was determined from cells treated with phagemid particles using the luminescent β-galactosidase analysis system from Clontech (Palo Alto, CA) according to the protocols suggested by the manufacturers. The cells were washed in PBS, cut into 1.5 ml microtubes, and protein concentration in the form of pellets was measured in the ready ones. The endogenous enzymatic activity was measured in the untransfected COS cell extract and subtracted from each reading. The β-galactosidase experiments were done in duplicate wells and repeated at least once.

In a normal experiment, 250-1800 (depending on the phage holder used, see the graph of dependent dose response in Figure 3A). The expression of GFP and autofluorescent cells are observed per well after 72 hours after the selection of FGF2 for COS-1 cells. As shown in Figure 3A, such as 107 of the phage (m.o.i of 240) resulting in the detection of cells expressing GFP. As shown by Figure 3B, more RC-CMVß phagemid particles were required to detect transgene activity, indicating that analysis of total cellular lystate ß-galactosidase was less sensitive than the detection of GFP-positive cells by direct observation. The results of several control experiments are described in Figures 4A-4C. When the increased amounts of FGF2 are used to compete with the selected phage junction, a 100-fold molar excess of FGF2 successively blocked in phagemid-dependent transfection of COS-1 cells (Figure 4A). The non-specific transduction observed with phages alone was not affected by the addition of the same amount of FGF2 (not shown). In addition, to investigate the possibility that transduction of cells by releasing the phage selection of FGF2 was dependent on the high affinity FGF receptor, the phage particles were selected in myoblast cells from rat L6, a cell line lacking high affinity FGF2 receptors (Olwin and Hauschka, J. Cell Biochem. 39: 443-454, 1989; available from ATCC; Manassas, VA) and compared with the response to establish the transfectant 16 (Flg37) constitutively overexpressing FGFR1 (obtained from Dr. Murray Korc, UCI, Irvine, CA; also easily created by those experienced in the art). As shown in Figure 4B. The presence of high affinity FGF2 facilitates the transfer of phage genes from FGF2 selection. A higher transduction efficiency is observed in the FgFR positive FgFR cells which are the L6 maternal cells and without GFP positive cells were observed in the cell line in the absence of NAV. A comparison between FGF2 selection and 2 different selection ligands, transferin was also carried out (Zatloukal et al., 1992) and EGF (Watkins et al., Gen Ther 4.J004-1012, 1997). Both are relatively inefficient in the release of pro-phage COS-1 cells for gene expression. The number of cells expressing GFP was markedly reduced when biotinylated FGF2 was replaced by biotinylated transferin or biotinylated EGF (Figure 4C). EXAMPLE 4 INTRACELLULAR TRAFFIC COS cells were treated with chloroquine as described in Sosnowski et al. (J. Biol. Chem. 277: 33647.33643, 1996), usually at concentrations of 50 to 100 μM for two hours. Sosnowski and others demonstrate a chloroquine potentiating effect when FGF2 selects the pure plasmid DNA, however, when chloroquine is used in combination with the transduction of the cells they maintain the phagemid particles that select FGF2, the chloroquine unexpectedly has an effect inhibitor on cell cell transduction (Figure 4D). EXAMPLE 5 FGF BLOCK FOR PHAGEMIDE PARTICLES USING POLYLYSIN In this example, FGF is linked to polylysine, which binds to the phagemid particles as a result of the interaction between the positively charged polylysine and the negatively charged phage. FGF is covalently linked to poly-D-lysine (average polymer length of 84) using S-2-pyridyl disulfide (SPDP) as described by Sosnowski et al. (J. Biol. Chem. 271: 33647-33653, nineteen ninety six). Phagemid EGFP-N1 particles were mixed with 5 μg of FGF-polylysine or 2.5 μg of polylysine alone (the FGF-polylysine was about 50% polylysine by weight) and was kept at room temperature for 30 minutes before the addition of COS cells that developed in 12-well plates. Cells were examined for 72 hours by fluorescent microscopy, and autofluorescent GFP-positive cell count. As shown in Figure 5, the GFP gene was translated into COS cells via the phages in present FGF-polylysine. The increased phage resulted in an increase in GFP positive cells. Approximately one third of the GFP positive cells represent the background due to non-specific phage absorption reversed with polylysine alone.

EXAMPLE 6 COVALENT FGF LINKAGE FOR PHAGEMIDE PARTICLES In this example, the coat proteins of the M13 phages were configured directly for FGF using heterobifunctional interlacing reagents. Free lysine in the termination N of the envelope protein of the Vil gene is available for the chimeric modification (Amstrong et al., EMBO J 2: 1641-1646, 1983). In this method, the phage particles were first treated with thiol by the addition of a 3-30 molar excess of SPDP in phosphate buffer pH 7.5, 0.1 M NaCI for 30 minutes at 23 ° C.

The unreacted reagent was removed by gel filtration, the FGF2-3 mutein of FGF was then reacted with the thiolated phage in molar excess 2-10 times for 24 hours at 23 ° C. The free FGF2-3 was removed by gel filtration. The bound phage for FGF was further purified by affinity chromatography on heparin-Sepharose column. The binding of FGF to the phage protein was configured by polyacrylamide gel electrophoresis and immunoassay of phage proteins. Under non-reducing conditions, the modified VG gene protein of FGF was changed from an apparent molecular mass of 5,500 Da to 23,500 Da. The addition of the reducing agent to the buffer disrupts the development of disulfide ligation and the yields of FGF (approximately 18,000 Da) and the protein of gene VIII.

Chemically modified FGF phages were analyzed for the ability to translate mammalian cells. The modified pEGFP-N1 phagemid particles of FGF delay the CMV-GFP expression cassette that was added to the COS cells seeded in 20,000 cells / well plates in 12-well plates. The cells were analyzed for GFP expression by fluorescence microscopy 72 hours after the addition of the phages. EXAMPLE 7 GENETIC FUSION OF FGF FOR PROTEIN WITH FAGOS COVERAGE In the following examples, a phage that inhibits FGF2 on its used surface binds to the FGF2 receptor on mammalian cells and is internalized. An FGF2 gene was subcloned into the type 3 vector of the modified M13 phage, MEGFP3 (Figure 1A), to create the ligand that exhibits the phage, MF2 / 1G3 (see Figure 1B). The MEGFP3 vector has been modified with the mammalian expression cassette to express the GFP of the reporter gene to monitor the transduction of mammalian cells by phages. Transduction results can be quantified using flow cytometry (FACscan, Becton Dickinson, Mountain View, CA) to measure the distribution of GFP expressing cells in the population and by fluorometry of cell extracts to measure the total product GFP. Other vectors include pCANTAB 5E (Pharmacia Biotech, Piscataway, NJ) or M13 type 3 or 33 for lll gene fusions (see Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, 1996, McConnell et al. , Mol.

Divers. 7: 165-176, 1996). Similarly, FGF2-3 was cloned into the M13 type 8 or 88 vector for the fusion of the gene VIII protein (Roberts et al., Methods Enzymol, 267: 68-82, 1996, Markland et al., Gen 709: 13-19 , 1991). To facilitate cloning. The FGF2 gene was amplified by PCR using the oligonucleotide primers containing the appropriate restriction endonuclease sites in the III or VIII genes of the phage vector. The resulting phages express FGF2 on their coated surface as detected by the anti-FGF2 antibodies in the Western blot analysis (Figure 6) and by ELISA (Figure 7). Binding of the FGF2 fusion phage to the FGF2 receptor was analyzed by ELISA in which the recombinant FGF2 receptor was bound to a solid phase and an antibody from the phage was used as the primary detection antibody. The detection by Western blot analysis of the fusion of FGF2-pIII uses the equivalent phage extracts of the purified FGF2 phage and the control phage (MEGFP3) separately by polyacrylamide gel electrophoresis and analyzes the stains in nitrocellulose. The phage-FGF2 and fusion of FGF2 was detected with the anti-FGF2 monoclonal antibody (Transduction Labs; Lexington, KY) and anti-mouse secondary antibody conjugated with HRP (American Qualex, San Clemente, CA) with chemiluminescent development. A single protein band was detected in purified cesium chloride from the purified FGF2 phage extract that migrate at approximately 80 kDa. Therefore approximately the predicted size for the fusion protein of FG F2-pl 11 (FGF2 (18 kDa) fused in plll (migrated approximately 60 kDa)). Purification of CsCl was carried out to remove any non-covalently bound FGF2 fusion protein from the phage particles. Binding of the FGF2 fusion phage to the FGF2 receptor was analyzed by ELISA in which the recombinant FGF2 receptor was bound to the solid phase and an anti-phage antibody was used as the primary detection antibody. Briefly, the phage was compared with an anti-FGF2 rabbit polyclonal antiserum bound to the well plate, an anti-M13 antibody conjugated to HRP (Pharmacia Biotech, Piscataway, NJ) was used to detect phage binding, when the antibody Anti-phage was used to capture the phage and the equivalent of OD was observed for the control of (MEGFP3) and the phage FGF2 (MF2 / 1G3) indicated that the equivalent phage particles are applied on the plate (Figure 7A). In Figure 7B an increased OD indicates the presence of FGF2 on the phage-FGF2 of MF2 / 1G3. EXAMPLE 8 TRANSFER OF MAMMALI CELLS BY THE FAGOS THAT EXHIBITS THE FGF LIGAND The phages exhibited by FGF2 (MF2 / 1G3) and identical phages lacking the FGF2 gene (MEGFP3) were compared for phage-mediated internment and reporter gene expression in COS cells. The phages were incubated with the cells for 4 hours at 37 ° C in DME (Dulbecco's Modified Eagle Medium, Sigma Chemical, St. Louis, MO) containing 2% BSA (bovine serum albumin) as a blocking agent. After washing to remove phages without binding the cells were returned to the incubator for an additional 72 hours. Transduction was measured by counting GFP-positive autofluorescent cells. As shown in Figure 8B, the phage exhibiting FGF2 resulted in approximately 10 times more transduction efficiency than the control phage initiating the FGF2 ligand exhibited on the surface of the phage particles resulting in the binding mediated by the receptor and the internment of the phages with the subsequent expression of the phage reporter gene. The specificity of the transduction measured by the FGF2 phage is demonstrated by the successive inhibition of transduction with free FGF2 excess (2 μg / ml) (Figure 8B). The lower level of non-specific absorption transduction by the control phage (MEGFP3) is not affected by the presence of excess FGF2. It is important to show that the control phage MEGFP3 is equally capable of translating the mammalian cells that display the phage when properly selected. To compare the transduction capacity of both phage-FGF2 and control phage, each phage-equipped was used to transfect COS cells using the avidin-biotin FGF2 selection method. In this method the biotinylated FGF2 can be contacted with the cells and used to capture the phage particles via the addition of avidin and a biotinylated anti-phage antibody. The phage / FGF2 / cell junction was carried out on ice, unbound phages were removed by washing, cells returned to the incubator at 37 ° C, and transduction was evaluated at 72 hours. As seen in Figure 8A, there is no significant difference in the transduction between FGF2-phage and the control phage when FGF2 bound to the phage towards an avidin-biotin ligation. In this case biotinylated FGF2 is an excess of FGF2 exhibited on the surface of the phages so that internment is expected to be mainly via the biotinylated FGF2. These data demonstrate the transduction measured by the specific receptor of mammalian cells by filamentous phages that genetically exhibits a selection ligand (FGF2). EXAMPLE 9 EXPRESSION SUSTAINED BY SELECTIVE PRESSURE In order to determine the expression of the transgene can be maintained in the cells after the introduction of the phagemid particles into the cells, resistance to neomycin was used to select the transfected cells. The cells were treated with the phagemid particles as described above and 48 hours later the cells were assembled in a ratio of 1:10 in fresh medium. The medium was removed and replaced with the selection medium containing G418 (70 μg / ml) 24 hours later. The colonies that survived formed (7-10 days), their GFP content was evaluated using fluorescence microscopy. The cell lines derived from these colonies were kept under selection of G418 (> 3 months). As shown in Figure 9A (100X magnification), when the translated cells were cultured in the presence of G418, the drug-resistant colonies appeared close to all cells that were positive for GFP activity. The colonies were expanded and maintained as stable cell lines expressing the GFP protein for 3 months (Figure 4B (400x magnification)). It should be appreciated that, while the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

Claims (45)

1. CLAIMS 1. A method for delivering genes, comprising: administering to a patient filamentous particles having a ligand on their surfaces, wherein the vector within the phage encodes a gene product under the control of a promoter. A method for treating tumors, comprising administering a pharmaceutical composition to a patient comprising a physiologically acceptable pH-regulating solution and filamentous phage particles having a ligament on their surfaces, wherein the phage genome encodes a gene product Therapeutics under the control of a promoter. 3. A method for treating smooth muscle diseases, comprising administering a pharmaceutical composition to a patient comprising a physiologically acceptable pH regulatory solution and filamentous phage particles having a ligand on their surfaces, wherein the phage genome encodes a product of therapeutic genes under the control of a promoter. 4. A method for treating angiogenic diseases, comprising administering a pharmaceutical composition to a patient comprising a physiologically acceptable pH regulatory solution and filamentous phage particles having a ligand on their surfaces, wherein the phage genome encodes a product of therapeutic genes under the control of a promoter. . t «> . t .. . , mm *? ? .. ...... t, r ... ». ^. - r. ...... ......... ^,. . . i aafefe »» a «ia > 5. The method according to any of claims 1-4, wherein the ligand is a polypeptide reactive with the FGF receptor. 6. The method according to claim 5, wherein the ligand is FGF-2. 7. The method according to any of claims 1-4, wherein the ligand is an antibody. 8. The method according to claim 7, wherein the antibody is a single chain antibody. 9. The method according to claim 7, wherein the antibody reacts with HER2 / neu. 10. The method according to any of claims 1-4, wherein the ligand is genetically fused with a phage capsid protein. 11. The method according to claim 10, wherein the capsid phage protein is the III gene. The method according to claim 10, wherein the phage capsid protein is gene VIII. The method according to any of claims 1-4, wherein the ligand is chemically conjugated with a phage capsid protein. The method according to any of claims 1-4, wherein the ligand further comprises an endosomal escape portion. __ i ^^^ ¿i «i-¿_ ^^^^ 15. The method according to any of claims 1-4, wherein the phage particles exhibit an endosomal escape peptide portion on the surface of the phage. 16. The method according to any of claims 1-4, wherein the phage genome is a phagemid. 17. The method according to any of claims 1-4, wherein the ligand further comprises a nuclear localization sequence. 18. The method according to any of claims 1-4, wherein the phage particles exhibit a nuclear localization sequence on the surface of the phage. 19. The method according to any of claims 1-4, wherein the product of therapeutic genes are selected from the group consisting of protein, ribozyme and antisense oligonucleotide 20. The method according to claim 19, wherein the Protein replaces a defective gene or alleviates a deficiency of the gene product. 21. The method according to any of claims 1-4, wherein the therapeutic gene product is a cytotoxic agent. 22. The method according to claim 21, wherein the cytotoxic agent is a ribosome that inactivates the protein. ^ yjgágg ^^^^^^ »^^ | ^« ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^ * ^ ggfe * fe ^^^ 23. The method according to claim 21, wherein the ribosome that inactivates the protein is saporin. 24. The method according to any of claims 1-4, wherein the therapeutic gene product is an antibody that binds HER2 / neu. 25. A pharmaceutical composition comprising a physiologically acceptable pH regulatory solution and the filamentous phage particles having a ligand on their surfaces, wherein the phage genome encodes a therapeutic gene product under the control of a promoter. 26. The composition according to claim 25, wherein the ligand is a polypeptide reactive with the FGF receptor. 27. The composition according to claim 26, wherein the polypeptide is FGF-2. 28. The composition according to claim 25, wherein the ligand is an antibody. 29. The composition according to claim 28, wherein the antibody is a single chain antibody. 30. The composition according to claim 28, wherein the antibody reacts with HER2 / neu. 31. The composition according to claim 25, wherein the ligand is genetically fused with a phage capsid protein. 32. The composition according to claim 31, wherein the phage capsid protein is the III gene. 33. The composition according to claim 31, wherein the phage capsid protein is gene VIII. 34. The composition according to claim 25, wherein the ligand is chemically conjugated to the phage capsid protein. 35. The composition according to claim 25, wherein the ligand further comprises an endosomal escape portion. 36. The composition according to claim 25, wherein the particles exhibit a portion of endosomal escape peptide on the surface of the phage. 37. The composition according to claim 25, wherein the bundle genome is a phagemid. 38. The composition according to claim 25, wherein the ligand further comprises a nuclear localization sequence. 39. The composition according to claim 25, wherein the phage particles exhibit a nuclear localization sequence on the surface of the phage. 40. The composition according to claim 25, wherein the therapeutic gene product is selected from the group consisting of protein, ribozyme, and antisense oligonucleotide. 41. The composition according to claim 25, wherein the protein replaces a defective gene or alleviates a deficiency of gene product. 42. The composition according to claim 25, wherein the therapeutic gene product is a cytotoxic agent. 43. The composition according to claim 42, wherein the cytotoxic agent is a ribosome that inactivates the protein. 44. The composition according to claim 42, wherein the ribosome that inactivates the protein is saporin. 45. Filamentous phage particles having a ligand on their surfaces, wherein the phage genome encodes a therapeutic gene product under the control of a promoter. (54) Title: SUPPLY OF GENES MEDIATED BY RECEIVERS USING BACTERIÓFAGOS VECTORS (57) SUMMARY Filamentous phage particles that exude a ligand on their surface are used to deliver a therapeutic gene in a cell. The ligand is a fusion protein with the protein of phage capsids, conjugated covalently in the particle particles, or complexed with the modified phage particles. ^ ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡J ^