MXPA98004203A - Vectors and methods for gene transfer acelu - Google Patents

Abstract

The present invention provides a chimeric adenovirus coat protein, which differs from the wild-type coat protein by the introduction of a non-native amino acid sequence. This chimeric adenovirus coat protein, according to the invention, is capable of directing the entry, into cells, of a vector comprising the coat protein, this entry is more efficient than the entry into cells of a vector that is identical, except that it comprises a wild-type adenovirus coat protein in place of the chimeric adenovirus coat protein. The chimeric coat protein is preferably a fiber, exon or penton protein. The present invention also provides an adenoviral vector comprising the chimeric coat protein, as well as methods for constructing and using this vect.

Description

VECTORS AND METHODS FOR GENE TRANSFER TO CELLS TECHNICAL FIELD OF THE INVENTION The present invention relates to a chimeric adenovirus coat protein that is capable of directing the entry into cells of a vector comprising the coat protein, which is more efficient than a similar vector having a protein. natural adenovirus cover. This chimeric coat protein is a protein fiber, exon or penton. The present invention also relates to a recombinant vector comprising this chimeric, adenoviral coat protein and methods for constructing and using this vector.

BACKGROUND OF THE INVENTION Adenoviruses corresponding to the Adenoviridae family, which is divided into two genera, specifically Mastadenovirus and Aviadenovirus. Adenoviruses are regular, unenveloped icosahedrons approximately 65 to 80 nanometers in diameter (Horne et al., J. Mol. Biol., 1, 84-86 (1959)). The adenoviral capsid is composed of 252 capsomeres of which 240 are exons and 12 are pentons (Ginsbert et al., Virology, 28, 782-783 (1966)). The exons and pentons are derived from three different viral polypeptides (Maizel and P1340 / 98MX collaborators, Virology, 36, 115-125 (1968); eber et al., Virology, 76, 709-724 (1977)). The exon comprises three identical polypeptides of 967 amino acids each, specifically polypeptide II (Roberts et al., Science, 232, 1148-115 (1986)). The penton contains a penton base, which is attached to the capsid, and a fiber, which binds non-covalently to the penton base and protrudes therefrom. The fiber protein comprises three identical polypeptides of 582 amino acids each, specifically polypeptide IV. The adenovirus serotype 2 penton-based protein (Ad2) is a ring-shaped complex composed of five identical protein subunits, 571 amino acids each, specifically polypeptide III (Boudin et al., Virology, 92, 125-138 (1979)). Proteins IX, VI and Illa are also present in the adenoviral envelope and are thought to stabilize the viral capsid (Stewart et al., Cell, 67, 145-154 (1991); Stewart et al., EMBO J., 12 (7) , 2589-2599 (1993)). Adenoviruses once bound to a cell undergo receptor-mediated internalization in cell-bound endocytic vesicles coated with clathrin (Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al., Virology, 40, 462-477 (1970)). The virions that enter the cell undergo a disassembly P1340 / 98MX gradual, in which many of the viral structural proteins are imparted (Greber et al., Cell, 75, 477-486 (1993)). During the de-coating process, the viral particles cause the breakdown of the cell endosome by a pH-dependent mechanism (Fitzgerald et al., Cell, 32, 607-617 (1983)), which is still poorly understood. The viral particles are then transported to the nuclear pore complex of the cell (Dales et al., Virology, 56, 465-483 (1973)), where the viral genome enters the nucleus, thereby initiating infection. To efficiently attack and infect a cell an adenovirus uses two separate cellular receptors, both of which must be present, (Wickham et al., Cell, 73, 309-319 (1993)). First, the Ad2 fiber protein binds the virus to a cell by binding to a receptor not yet identified. Then, the penton base binds to av integrins, which are a family of surface receptors of heterodimeric cells, which mediate cell adhesion to extracellular matrix molecules, as well as other molecules (Hynes, Cell, 69, 11-125 (1992)). The fiber protein is a trimer (Devaux et al., J. Molec, Biol., 215, 567-588 (1990) consisting of an extension, a tree and a projection.

P1340 / 98MX fiber tree region is composed of repeating 15 amino acid portions, which is thought to form two alternating ß-strands and ß-curves (Green et al., EMBO J, 2, 1357-1365 (1983).) The total length of the fiber tree region and number of 15 amino acid repeats They differ between adenoviral serotypes For example, the Ad2 fiber tree is 37 nanometers long and contains 22 repeats, while the Ad3 fiber is 11 nanometers long and contains 6 repeats. of the fiber protein is located in the region of the projection encoded by the last 200 amino acids of the protein (Henry et al., J. Virology, 68 (8), 5239-5246 (1994)). The regions necessary for trimerization are also located in the region of the protein overhang (Henry et al. (1994), supra). A deletion mutant lacking the last 40 amino acids does not trimerize and also does not bind to the penton base (Novelli et al., Virology, 185, 365-376 (1991)). In this way, trimerization of the fiber protein is necessary for the union of the penton base. The nuclear localization signals that direct the protein to the nucleus to form the viral particles that follow its synthesis in the cytoplasm are located in the N-terminal region of the protein (Novelli et al. (1991), supra). The P1340 / 98 X fiber, together with the exon, are the main antigenic determinants of the virus and also determine the specificity of the virus serotype (Atson et al., J. Gen. Virol, 69, 525-535 (1988)). For the directed transfer to cells of one or more recombinant genes to diseased cells or diseased tissue in need of treatment, recombinant adenoviral vectors have been used. These vectors are characterized by the advantage that they do not require the proliferation of host cells for the expression of the adenoviral proteins (Horwitz et al., In Virology, Raven Press, New York, Vol. 2, pp. 1679-1721 (1990); and Berkner, BioTechniques, 6, 616 (1988)). Furthermore, if the tissue assigned for somatic gene therapy is the lung, these vectors have the additional advantage of being normally trophic for the respiratory epithelium (Straus, In Adenoviruses, Plenan Press, New York, pp. 451-496 (1984)) . Other advantages of adenoviruses as potential vectors for human gene therapy are: (i) recombination is rarely observed with the use of these vectors; (ii) with adenoviral infections there are no known associations of human malignancies, despite human infection common with adenoviruses; (iii) you can manipulate the adenoviral genome (which is a DNA of P1340 / 98MX double strand, linear) to handle foreign genes that vary in size; (iv) an adenoviral vector does not insert its DNA into the chromosome of a cell, so that its effect is impermanent and unlikely to interfere with the normal function of the cell; (v) the adenoviruses can affect non-division and terminally differentiated cells, such as cells in the brain and lungs; and (vi) live adenoviruses, which have the ability to replicate as an essential characteristic, have been used safely as a vaccine in humans (Horwitz et al., (1990), supra; Berkner et al., (1988), supra). Straus et al., (1984), supra, Chanock et al, JAMA, 195, 151 (1966), Haj-Ahmad et al., J. Virol., 57, 267 (1986), and Ballay et al., EMBO, 4 , 3861 (1985), PCT patent application WO 94/17832) A disadvantage of adenovirus-mediated gene therapy is that after two weeks following administration of the vector, significant decreases in gene expression are observed. the loss of expression requires the re-administration of the viral vector, however, after re-administration, neutralizing antibodies are formulated against the proteins of both the fiber and the exon of the viral vector (Wohlfart, J. Viroloqv, 62, 2321-2328 (1988); Wohlfart et al. speakers, P1340 / 98MX J. Virology, 5_6, 896-903 (1985)). This antibody response against the virus can prevent the effective readministration of the viral vector. Certain cells are not easily treatable to the adenovirus-mediated gene distribution which is another disadvantage of using recombinant adenoviruses in gene therapy. For example, lymphocytes, which lack the adenovirus receptors of av integrin, are damaged in the incorporation of adenoviruses (Silver et al., Virology 165, 377-387 (1988); Horvath et al., J. Virology, 62 (1), 341-345 (1988). This lack of ability to infect all cells has led researchers to look for ways to introduce adenovirus into cells that can not be infected by adenovirus, for example, due to the lack of adenoviral receptors. In particular, the virus can be coupled to a DNA-polylysine complex containing a ligand (e.g., transferrin) for mammalian cells (e.g., Wagner et al., Proc. Nati. Acad. Sci., 8_9, 6099 -6103 (1992); PCT Patent Application WO 95/26412). Similarly, the adenoviral fiber protein can be spherically blocked with antibodies, and tissue-specific antibodies can be chemically linked to the viral particle (Cotten et al, Proc Nati Acad Sci USA, 89, 6094-6098 P1340 / 98MX (1992)). However, these approaches are disadvantageous because they require additional steps that link covalently the virus large molecules, such as polylysine, receptor ligands and antibodies (Cotten (1992), supra;.... Wagner et al, Proc Natl Acad Sci, 89, 6099-6103 (1992)). This is added to the size of the resulting vector as well as to its production cost. In addition, the assigned particle complexes are not homogeneous in their structure, and their efficiency is sensitive to the relative relationships of the viral particles, binding molecules, and target selection molecules used. In this way, this approach is less optimal to extend the repertoire of treatable cells to adenovirus-mediated gene therapy. Recently, it has questioned the efficiency of gene transfer in vivo mediated by adenovirus even those cells which adenovirus has been estimated that enter with high efficiency (Grubb et al, Nature 371, 802-806 (1994); and Dupuit collaborators, Human Gene Therapy, 6, 1185-1193 (1995)). The fiber receptor by means of which the adenovirus makes initial contact with the cell has not been identified and its representation in different tissues has not been examined. It is generally assumed that epithelial cells P1340 / 98MX in the lung and intestine produce sufficient fiber receptor levels to allow optimal transduction. However, no study has confirmed this point to date. In fact, studies have suggested that the distribution of adenovirus genes to undifferentiated lung epithelium is less efficient than distribution to proliferating or undifferentiated cells (Grubb et al., Supra, Dupuit et al., Supra). Similarly, it has been shown that adenoviruses transduce a large number of tissues including lung epithelial cells (Rosenfeld et al, Cell, 68, 143-155 (1992)), muscle cells (Quantin et al, Proc. Nati. Acad. Sci., 89, 2581-2584 (1992)), endothelial cells (Lemarchand et al, Proc. Natl. Acad. Sci., 89, 6482-6486 (1992), fibroblasts (Anton et al, J. Virol, 69, 4600-4606 (1995)), and neuronal cells (LaSalle et al., Science, 259, 988-990 (1993)). However, in many of these studies, very high levels of virus particles have been used to achieve transduction, often exceeding 100 that plaque forming units (pfu) / cell, and corresponding to a multiplicity of infection (MOI) of 100. the requirement for a high MOI to achieve transduction is disadvantageous, as any immune response associated with the adenoviral infection will necessarily be exacerbated P1340 / 98MX with use of high doses. Accordingly, there still remains a need for vectors, such as adenoviral vectors, which are capable of infecting cells with high efficiency, especially at lower MOI, and which demonstrate an increased range of host cells of infectivity. The present invention seeks to overcome at least some of the aforementioned problems of recombinant adenoviral gene therapy. In particular, it is an object of the present invention to provide a vector (such as an adenoviral vector) having a broad host range, and an ability to introduce cells at a high efficiency, at a reduced MOI, thereby reducing the amount administered from the recombinant adenoviral vector and any side effect / complication resulting from this administration. A further object of the present invention is to provide a method of gene therapy comprising the use of a homogeneous adenovirus, wherein the viral particle is modified at the level of the adenoviral genome, without the need for additional chemical modifications of the viral particles. These and other objects and advantages of the present invention, as well as inventive features, will be apparent from the following detailed description.

P1340 / 98MX BRIEF SUMMARY OF THE INVENTION The present invention provides a chimeric, adenoviral coat protein (e.g., a fiber, exon or penton protein), which differs from the native fiber (i.e., native) protein by introduction of a non-native amino acid sequence, preferably at or near the carboxyl terminus. The chimeric protein resulting from adenovirus coating is capable of directing the entry into cells of a vector comprising the coat protein that is more efficient than the entry into cells of a vector that is identical, except that it comprises a natural coat protein. adenovirus instead of chimeric adenovirus coat protein. A direct result of this increased input efficiency is that the chimeric adenovirus coat protein allows the adenovirus to bind and enter a number of cell types that the adenovirus, which comprises the native coat protein, typically can not enter. or can enter with only high efficiency. The present invention also provides an adenoviral vector comprising the chimeric adenovirus coat protein, and methods for constructing and using this vector.

P1340 / 98MX BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a bar graph depicting the binding (percent entry) of natural adenovirus to cells derived from different tissues. Figures 2A-B depict the binding of a nucleic acid sequence at the end of the natural adenoviral fiber gene (Figure 2A) to derive a chimeric, adenoviral fiber protein (Figure 2B) comprising a non-native sequence of amino acids in the carboxy term. As indicated, the length of the polyA prolongation, and consequently, the number of Usins in the resulting protein, can vary. Figure 3 is a schematic diagram representing the construction of the adenovirus transfer vector containing the chimeric fiber protein pAd BS 59-100 UTV by means of intermediate transfer vectors. In particular, pAd NS 83-100 (also known as pl93NS 83-100 or pNS 83-100) is used to derive less fiber (ie F '') pAd NS 83-100 (also known as pl93NS (? F) or pNS (? F)) (path A), pAd NS 83 100 (F ") is used to derive pAd NS 83-100 UTV (also known as pl93NS (F5 *), pl93 (F5 *), or pNS (F5) *)) (path B), and pAd NS 83-100 UTV is used to derive pAd BS 59-100 UTV (path C) Figures 4A-D represent oligonucleotides P1340 / 98MX used for the construction of GV10 UTV, ie the primers SEQ ID NO: 9 (Figure 4A), SEQ ID NO: 10 (Figure 4B), SEQ ID NO: 11 (Figure 4C), and SEQ ID NO : 12 (Figure 4D). Figure 5 depicts a Western blot showing the increase in size of the fiber adenoviral chimeric protein (UTV) compared to the natural fiber (WT) protein. Figures 6A-B are graphs depicting a comparison of the binding of an adenoviral vector comprising the natural fiber protein (i.e., GV10, open triangle) and the adenoviral vector comprising the chimeric fiber protein (i.e., GV10 UTV, filled circles) to a receptor-plus cell (A549, Figure 6A) and receptor-minus (HS 68, Figure 6B). Figure 7 is a graph of UTV binding (counts per minute (CPM)) against competitor amount (μg / ml) for the inhibition of fiber adenoviral chimeric protein binding to receptor-minus cells (ie, HS 68 fibroblasts) by the soluble factors, chondroitin sulfate (open circle); heparin (filled circle); mucin (filled triangle); and salmon sperm DNA (open triangle). Figure 8 is a graph of the binding of UTV (CPM) against enzyme dilution for inhibition of P1340 / 98MX binding of chimeric adenoviral fiber protein to receptor-minus cells (ie, HS 68 fibroblasts) by enzymes, chondroitinase (open circles, dotted lines); heparinase (open circles, solid lines); and sialidase (triangles, solid lines). Figure 9 is a bar chart depicting a comparison of the transfer of a lacZ reporter gene to an adenoviral vector comprising the natural fiber protein (i.e., GV10) and an adenoviral vector comprising the chimeric fiber protein (e.g. say, GV10 UTV) as assessed by the expression of the resulting reporter gene (ie, relative light units (RLU)) in several receptor-plus and receptor-minus cells. Figure 10 is a bar chart depicting a comparison of the transfer of a lacZ reporter gene to an adenoviral vector comprising the natural fiber protein (i.e., GV10) and an adenoviral vector comprising the chimeric fiber protein (e.g. say, GV10 UTV) as assessed by the resulting indicator expression (ie, relative light units (RLU) in the mouse lung.) Figure 11 is a bar graph depicting the transfer of a reporter gene (i.e. in pGUS) by an adenoviral vector comprising the natural fiber protein (ie, GV10, solid bar) P1340 / 98MX and an adenoviral vector comprising chimeric fiber (ie, GV10 UTV, open bars) potentially opened via a protein / DNA interaction in 293 cells, A549 cells and H700 T cells. Figure 12 is a diagram that further represents the plasmid pl93 (F5 *) (described as pAd NS 83-100 UTV in Figure 3, and also known as pl93 (F5 *) or pNS (F5 *)) used to construct the adenovirus fiber chimeras, and the sequence of the C-terminus of the mutated fiber protein present from the plasmid (animated polyadenylation site). Figure 13 is a diagram representing the plasmid pl93NS (F5 *) pGS (K7) (also known as pl93 (F5 *) pGS (K7) or pNS (F5 *) pK7) used to construct adenovirus chimeras. Figure 14 is a diagram representing the pBSS 75-100 pGS (null) plasmid (also known as pBSS 75-100? E3 pGS (null)). Figure 15 is a diagram representing the pBSS 75-100 pGS (RK32) plasmid (also known as pBSS 75-100? E3 pGS (RKKK), or pBSS 75-100? E3 pGS (RKKK2)). Figure 16 is a diagram representing plasmid pBSS 75-100 pGS (RK33) (also known as pBSS 75-100? E3 pGS (RKKK), or pBSS 75-100? E3 pGS (RKKK3)). Figure 17 is a diagram representing the P1340 / 98MX plasmid pl93NS F5F2K (also known as pl93 F5F2K). Figure 18 is a diagram representing the plasmid pl93NS F5F2K (RKKK) 2 (also known as pl93NS F5F2K (RKKK2), pl93NS F5F2K (RK32), or pl93 F5F2K (RKKK2)). Figure 19 is a diagram representing the plasmid pl93NS F5F2K (RKKK) 3 (also known as pl93NS F5F2K (RKKK3), pl93 F5F2K (RKKK3), or pl93 F5FK (RK33)). Figure 20 is a diagram depicting plasmid pACT (RKKK) 3 (also known as pACT (RKKK3) or pACT (RK33)). Figure 21 is a diagram representing the pACT (RKKK) 2 plasmid (also known as pACT (RKKK2) or pACT (RK32)). Figure 22 is a diagram representing the plasmid pACT Hll. Figure 23 is a diagram depicting plasmid pACT H11 (RKKK) 2 (also known as pACT H11 (RKKK2) or pACT H11 (RK32)). Figure 24 is a diagram representing the pl93 plasmid F5F9sk (also known as pl93 F5F9K-short). Figure 25 is a diagram representing the plasmid pSPdelta. Figure 26 is a diagram representing the plasmid pSP2alpha. The "j" indicates the PpulOI sites P1340 / 98MX destroyed in the plasmid. Figure 27 is a diagram representing the plasmid pSP2alpha2. The "j" indicates the PpulOI sites destroyed in the plasmid. Figure 28 is a plot of the post-infection days against FFU / cell for 293 cells infected with Ad5 (open circles AdZ.F (RGD) (closed squares), or AdZ.F (pK7) (open triangles).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides, inter alia, a recombinant adenovirus comprising a chimeric coat protein, such as a chimeric fiber, penton and / or exon protein. The chimeric coat protein comprises a non-native amino acid sequence, in addition to, or instead of, a native amino acid sequence. This non-native sequence of amino acids allows the chimeric fiber (or a vector comprising the chimeric fiber) to bind more efficiently to and enter cells. Thus, the present invention provides a chimeric adenovirus coat protein comprising a non-native amino acid sequence, wherein the chimeric protein is capable of directly entering cells of a vector comprising the P1340 / 98MX coat protein that is more efficient than the entry into cells of a vector that is identical, except that it comprises a native adenovirus coat protein in place of the chimeric adenovirus coat protein (i.e., in the absence of the chimeric adenovirus coat protein and in the presence of the native adenovirus coat protein).

Chimeric Cover Protein A "cover protein" according to the invention preferably comprises a fiber protein (especially, an adenoviral fiber protein), a penton protein (especially a penton adenoviral protein), and an exon protein (especially an exon adenoviral protein). In particular, a coat protein preferably comprises a fiber, penton or exon adenoviral protein. Any of the human or non-human adenovirus serotypes can be used as the source of the coat protein gene, optimally, however, the adenovirus is an Ad2 or Ad5 adenovirus. The coat protein is "chimeric" in that it comprises amino acid residues that are not typically found in the protein as it is isolated from the natural adenovirus (i.e., comprising the protein Native P1340 / 98MX, or natural protein). In this manner, the coat protein comprises a "non-native amino acid sequence". By "non-native amino acid sequence" is meant any amino acid sequence that is not found in the native fiber of a given adenovirus serotype that is preferentially introduced into the fiber protein at the level of gene expression (i.e. , by the introduction of a "nucleic acid sequence encoding a non-native amino acid sequence"). This non-native sequence of amino acids comprises an amino acid sequence (i.e., has component residues in a particular order) that imparts an ability to bind to and enter cells in the resulting chimeric protein through a new binding site to the cell surface (ie, a "UTV sequence", or "Universal Transfer Vector sequence"), and / or comprising a sequence incorporated to produce or maintain a certain configuration of the resulting chimeric protein (i.e. a "spawning sequence") between the native / non-native, non-native / non-native, or native / native sequence. Since the non-native sequence of amino acids is inserted into or in place of an amino acid sequence, and the manipulation of the amino acid sequence of the chimeric coat protein is done P1340 / 98MX preferably at the level of the nucleic acid, the amino acid sequence that differs in the chimeric coat protein, from the natural coat protein (i.e., the UTV sequence and the spawning sequence) can preferably comprise a sequence of completely non-native amino acids, or a mixture of native and non-native amino acids). A "cell surface binding site" encompasses a receptor (which preferably is a protein, carbohydrate, glycoprotein, or proteoglycan) as well as any molecule charged in an opposite manner (ie, charged in an opposite manner with respect to the chimeric protein of cover, preferably comprising a non-native amino acid sequence that is positively charged, as further described herein) or another type of molecule with which the chimeric coat protein can interact to bind to the cell, and thereby promote the entrance into the cell. Examples of potential cell surface binding sites include, but are not limited to: negatively charged heparin, heparan sulfate, hyaluronic acid, dermatan sulfate, and portions of chondroitin sulfate, for example, found in glycosaminoglycans , and which may also contain sialic acid, sulfate and / or phosphate, portions of sialic acid P1340 / 98MX found in mucins, glycoproteins and gangliosides; glycoproteins of the major histocompatibility complex I (MHC I); common components of carbohydrates found in membrane glycoproteins, including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, mucosa, galactose, and the like; and portions of phosphate, for example, in nucleic acids. However, a chimeric coat protein according to the invention, and methods of using it, is not limited to any particular mechanism of cell interaction (i.e., interaction with a particular cell surface binding site) and does not It should be considered in this way. Additionally, this cell surface binding site is "new" since the site is one that was previously inaccessible to the interaction with a cover adenoviral protein (i.e., the adenoviral, native coat protein such as fiber protein) , or was inaccessible only at a very low level, as reflected by the reduced efficiency of the entry of a vector containing the adenoviral, native coat protein compared to a vector comprising a chimeric adenovirus coat protein such as protein of fiber according to the invention. In addition, the union site is new since it is present in the majority P1340 / 98MX of the cells, if not all, despite their origin. This is in contrast to the cell binding site with which it is presumed to interact with the natural, fiber adenoviral protein that is ostensibly present only in a subset of cells, or is only accessible in a subset of cells, as reflected by the efficiency Reduced entry of adenoviral vector containing fiber, natural. The "input efficiency" can be quantified by several means. In particular, the input efficiency can be quantified by introducing the chimeric coat protein into a vector, preferably a viral vector, and inspecting the cell entry (eg, by vector-mediated distribution to a cell of a gene such as a gene). indicator) as a function of the multiplicity of infection (MOI)). In this case, a reduced MOI required for entry into cells of a vector comprising a chimeric adenoviral envelope protein as compared to a vector that is identical except that it comprises a natural envelope viral protein in place of the chimeric envelope protein of adenovirus, indicates a "more efficient" entry. Similarly, the input efficiency can be quantified in terms of the capacity of vectors containing chimeric, cover proteins, or P1340 / 98MX natural, or chimeric or natural, soluble cover proteins, to bind to cells. In this case, the increased binding exhibited by the vector containing a chimeric, adenoviral coat protein, or the chimeric coat protein itself, as compared to the identical vector containing a native coat protein in place of, or the protein of, natural cover itself, is indicative of increased input efficiency, or a "more efficient" input. A non-native sequence of amino acids according to the invention is preferably inserted into or instead of an internal coat protein sequence. Alternatively, a non-native amino acid sequence according to the invention is in or near the C-terminus of a protein. In particular, when a cover protein according to the invention is a fiber protein, desirably a non-native amino acid sequence is at or near the C-terminus of the protein. When a cover protein according to the invention is a penton or exon protein, preferably a non-native amino acid sequence is within an exposed loop of the protein, for example, as described in the following examples, particularly within of a hypervariable region in loop 1 and / or loop 2 of the adenovirus exon protein P1340 / 98MX (Crawford-Miksza et al., J. Virol., 70, 1836-1844 (1996)). Thus desirably a non-native amino acid sequence is in a region of a coat protein that is capable of interacting with and binding to the cell. Additionally, the method of the invention can be used to create adenoviral vectors containing UTV or UTV-like sequences in an extended (or perforated) structure, particularly in exon and / or penton base protein, to result in elongated proteins exon and / or penton base, wherein the amino acid insertion protrudes outward from the protein since it is present in a virion capsule. In particular, these perforated shell proteins can be incorporated into a recombinant adenovirus together with a "short tree fiber" (further described herein), wherein the fiber tree has been shortened, and optionally, the protrusion of the Fiber protein has been replaced with a projection (including the trimerization domain) of another serotype adenoviral vector from which the remaining part of the fiber protein is derived. Accordingly, the short tree fiber protein can be preferably incorporated into an adenovirus having a chimeric, penton-based protein that P1340 / 98 X comprises a sequence of UTV or similar to UTV, or having a "perforated" chimeric penton base protein that can optionally also incorporate a UTV or UTV-like sequence. Also, the short tree fiber protein can be incorporated into an adenovirus having a chimeric exon protein comprising a sequence of UTV or similar to UTV, or having a "perforated" chimeric exon protein that can optionally also incorporate a UTV sequence or similar to UTV. Optimally, the non-native sequence of amino acids is linked to the protein by another non-native amino acid sequence, ie, by a spacer, intermediate sequence. A spacer sequence is a sequence that intervenes between the native sequence of protein and the non-native sequence, between a non-native sequence and another non-native sequence, or between the native sequence and another native sequence. A spacer sequence is preferably incorporated into the protein to ensure that the non-native sequence comprising the cell surface binding site protrudes or projects from the three-dimensional structure of the chimeric protein (especially, the three-dimensional structure of the chimeric protein as it exists in nature, ie, as part of a capsid) in such a way as to be able to interact with and bind to the cells. When P1340 / 98MX inserts a spawning sequence in or replaces an internal coat protein sequence, one or more spawning sequences may be present in the chimeric coat protein. A spacer, intermediate sequence can be of any suitable length, preferably from about 3 to about 400 amino acids for a spacer sequence added to derive a "perforated" coat protein (as further described in the following examples), and preferably from about 3 to about 30 amino acids for any other application described herein. A spacer sequence can comprise any amino acid. Optimally, the spacer sequence does not interfere with the functioning of the coat protein in general, and in particular the functioning of the other non-native amino acid sequences, (ie, the UTV or UTV-like sequence). The non-native amino acid sequence is not a spacer sequence, ie, the UTV sequence can also be of any suitable length, preferably from about 3 to about 30 amino acids (although, optionally, as for the spacer sequence, the sequence of UTV may be longer, P1340 / 98MX example, up to 400 amino acids). These amino acids are preferably any of the positively charged residues that are capable of binding charged portions relatively present on the surface of a eukaryotic cell, and are optimally capable of binding to relatively charged portions that are present on the surface of most cells eukaryotic (if not all). In particular, this negatively charged portion present on the surface of a eukaryotic cell to which the UTV sequence binds includes the "cell surface binding site" mentioned above. Desirably, the non-native amino acid sequence comprises amino acids selected from the group consisting of lysine, arginine and histidine. Alternatively, these amino acids may be negatively charged residues that are capable of binding to the cell surface binding sites, positively charged, eg, desirably the non-native amino acid sequence comprising amino acids selected from the group consisting of of aspartate and glutamate. In this manner, the non-native amino acid sequence of a coat protein preferably comprises a sequence selected from the P1340 / 98MX group consisting of: SEQ ID NO: 1 (ie, Lys Lys Lys Lys Lys Lys Lys Lys), SEQ ID NO: 2 (ie Arg Arg Arg Arg Arg Arg Arg Arg), and SEQ ID NO: 3 (ie Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa), wherein "Xaa" comprises Lys or Arg), and wherein 1, 2, 3, 4, or 5 residue of the sequence can be deleted in the C-terminus Of the same. When the coat protein is a fiber protein, preferably the protein comprises a selected sequence consisting of the group of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, (ie, Gly Ser Asn Lys Glu Ser Phe Val Leu Lys Lys Lys Lys Lys Lys), and SEQ ID NO: 5 (ie Ala Gly Ser Asn Lys Asn Lys Glu Ser Phe Val Leu Lys Lys Lys Lys Lys Lys ), and where 1, 2, 3, 4, or 5 residues of the sequence can be deleted in the C-terminus thereof. Also, sequences that bind to heparin may be comprised in binding to a major heparin receptor (Sawitzky et al., Med. Microbiol, Immunol., 182, 285-92 (1993)). Similarly, so-called "heparin binding sequences" can mediate the interaction of the peptide or protein in which they are contained with other cell surface binding sites, such as cell surface heparan sulfate proteoglycan (Thompson et al. collaborators, J. Biol. Chem., 269, 2541-9 (1994)). In this way, the P1340 / 98MX non-native amino acid sequence (ie, the UTV sequence) preferably comprises these sequences, as well as additional sequences that are capable of recognizing a negatively charged portion, widely represented on the surface of eukaryotic cells. In particular, preferably, the non-native amino acid sequence comprises two basic amino acids (frequently Arg) located approximately 20A apart, giving in opposite directions of the alpha-helix (Margalit et al., J. Biol. Che., 268, 19228-31 (1993); Ma et al., J. Lipid Res., 35, 2049-2059 (1994)). Other basic amino acids are desirably dispersed between these two residues, giving one side, while the non-polar residues give the other side, forming an antipathetic structure, which optimally comprises the portion XBBXBX [SEQ ID NO: 49] or XBBBXXBX [SEQ ID NO: 50], wherein B is a basic amino acid (e.g., Lys, Arg, etc.), and X is any other amino acid. Also, preferably, the non-native amino acid sequence of UTV comprises: the sequence LIGRKKT [SEQ ID NO: 51], LIGRK (SEQ ID NO: 52) or LIGRR [SEQ ID NO: 53], which are common portions of heparin binding present in fibronectin and heat shock proteins (Hansen et al., Biochim, Biophys, Acta, 1252, 135-45 (1995)); the inserts of 7 waste already P1340 / 98MX is either Lys or Arg, or mixtures of Lys and Arg (Fromm et al., Arch. Biochem. Biophys., 323, 279-87 (1993)); the common basic C-terminal region of IGFBP-3 and IGFBP-5 of about 18 amino acids and comprising a heparin binding sequence (Booth et al., Growth Regul., 5, 1-17 (1995)); either one or both of the two hyaluronan binding portions (HA) located within a 35 amino acid region of the C-terminus of the HA receptor, RHAMM (Yang et al., JL Cell. Biochem., 56, 455-68 (1994)); a synthetic peptide (Ala347-Arg361) modeled after the heparin-binding form of Staphylococcus aureus vitronectin comprising heparin-binding consensus sequences (Liang et al., J. Biochem., 116, 457-63 (1994) ); any or more than five heparin binding sites between amino acid 129 and 310 of the gilí glycoprotein of bovine hypervirus 1 or any of four heparin binding sites between amino acids 90 and 275 of the glycoprotein gilí of pseudorabies virus (Liang and collaborators, Virol., 194, 233-43 (1993)); amino acids 134 to 141 of the glycoprotein gilí of pseudorabies viruses (Sawitzky et al., Med. Microbiol. Immunol., 182, 285-92 (1993)); the heparin binding regions corresponding to the charged residues at positions 279-282 and 292-304 of lipoprotein lipase (Ma P1340 / 98MX et al., Supra); a 22-residue, synthetic peptide, N22W, with a sequence NVSPPRRARVTDATETTITISW [SEQ ID NO: 54] or residues TETTITIS [SEQ ID NO: 55] of this synthetic peptide modeled after fibronectin and exhibiting heparin binding properties ( Ingham et al., Arch. Biochem. Biophys., 314, 242-246 (1994)); the GVEFVCCP portion [SEQ ID NO: 56] present in the ectodomain zinc binding site of Alzheimer's beta-amyloid precursor protein (APP), as well as several other similar APP proteins, which modulate the affinity to heparin ( Bush et al., J. Biol. Chem., 229, 26618-21 (1994)); Peptides of 8 amino acid residues derived from the inter-region of the laminin A chain (Tashiro et al., Biochem, J _, 302, 73-9 (1994)); synthetic peptides based on the heparin-binding region of antithrombin III from inhibitor to serine protease including the peptides F123-G148 and K121-A134 (Tyler-Cross et al., Protein Sci., 3, 620-7 ( 1994)); a 14 K N-terminal fragment of APP and a region near the N-terminus (ie, residues 96-110) proposed as the heparin binding regions (Small et al., J. Neurosci., 14, 2117-27 (1994)); and a 21-amino acid stretch of the growth factor, similar to the heparin-binding epidermal growth factor (HB-EGF) characterized with a high content of P1340 / 98MX lysine and arginine residues (Thompson et al., J. Biol. Chem., 269, 2541-9 (1994)); a region of 17 amino acids comprising the heparin region of thrombospondin and corresponding to a synthetic peptide of hep 1 (Murphy-Ullrich et al., J. Biol. Chem., 268, 26784-9 (1993)); a 23 amino acid sequence (Y565-A587) of the human von Willebrand factor that binds to heparin (Tyler-Cross et al., Arch, Biochem. Biophys., 306, 528-33 (1993)); and the PRARI peptide derived from fibronectin [SEQ ID NO: 57] and larger peptides comprising this portion, such as WQPPRARI [SEQ ID NO: 58]) that binds to heparin (Woods et al., Mol. Biol. Cell. , 4_, 605-613 (1993), and the heparin-binding region of platelet factor 4 (Sato et al., Jpn. J. Cancer Res., 84, 485-8 (1993), and the K18K sequence in the tyrosine transaminase glycoprotein of the fibroblast growth factor receptor (Kan et al., Science, 259, 1918-21 (1993).) In addition, the UTV sequence can comprise other sequences that are described in the following examples In this way, preferably the UTV sequence is selected from the group consisting of: [SEQ ID NO: 1], [SEQ ID NO: 2], [SEQ ID NO: 3], [SEQ ID NO. : 4], [SEQ ID NO: 5], [SEQ ID NO: 20], [SEQ ID NO: 22], [SEQ ID NO: 24], [SEQ ID NO: 26], [SEQ ID NO: 28] ], [SEQ ID NO: P1340 / 98MX 30], [SEQ ID NO: 32], [SEQ ID NO: 34], [SEQ ID NO: 36], [SEQ ID NO: 38], [SEQ ID NO: 40], [SEQ ID NO. : 42], [SEQ ID NO: 46], [SEQ ID NO: 48], [SEQ ID NO: 49], [SEQ ID NO: 50], [SEQ ID NO: 51], [SEQ ID NO: 52 ], [SEQ ID NO: 53], [SEQ ID NO: 54], [SEQ ID NO: 55], [SEQ ID NO: 56], [SEQ ID NO: 57], [SEQ ID NO: 58], [SEQ ID NO: 73], [SEQ ID NO: 74], [SEQ ID NO: 76], [SEQ ID NO: 78], [SEQ ID NO: 90] and [SEQ ID NO: 93]. These sequences can also be used where 1, 2, or 3 of the sequences are deleted in the C- or N-terminus. Also, since a spacer sequence can be any amino acid sequence that does not interfere with the functioning of the protein, according to the invention, any of the UTV sequences mentioned above can also comprise spacer sequences. It is also preferable that the non-native amino acid sequence comprises amino acid sequences that are "equivalent" to any of the sequences mentioned above (ie, they are "UTV-like sequences"). An equivalent can be a sequence that has the same function (with perhaps less differences in effectiveness) and still differs slightly in terms of its amino acid sequence, or other structural characteristics. In particular, an equivalent sequence is one comprising one or more conservative amino acid substitutions of the P1340 / 98MX sequence. An "amino acid conservative substitution" is an amino acid substituted by an alternative amino acid of similar charge density, hydrophilicity / hydrophobicity, size, and / or configuration (eg, Val per lie). In comparison, a "non-conservative substitution of amino acids" is an amino acid substituted by an alternative amino acid of different charge density, and hydrophilicity / hydrophobicity, size and / or configuration (eg, Val by Phe).

Nucleic Acid Coding for a Chimeric Coated Protein As previously indicated, preferably the non-native amino acid sequence is introduced at the DNA level. Accordingly, the invention preferably provides an isolated and purified nucleic acid encoding a coat protein according to the invention, wherein the nucleic acid sequence encoding the non-native amino acid sequence comprises a sequence of SEQ ID NO: 6 (ie, GGA TCC AA), which is located before the polyadenylation site. Similarly, the invention preferably provides an isolated and purified nucleic acid comprising a sequence selected from the group consisting of: SEQ ID NO: 7 (ie, P1340 / 98MX GGA TCC AAT AAA GAA TCG TTT GTG TTA TGT) and SEQ ID NO: 8 (ie, GCC GGA TCC AAC AAG AAA GAA TCG TTT TGT TTA), [SEQ ID NO: 19], [SEQ ID NO. : 21], [SEQ ID NO: 23], [SEQ ID NO: 25], [SEQ ID NO: 27], [SEQ ID NO: 29], [SEQ ID NO: 31], [SEQ ID NO: 33] ], [SEQ ID NO: 35], [SEQ ID NO: 37], [SEQ ID NO: 39], [SEQ ID NO: 41], [SEQ ID NO: 45], [SEQ ID NO: 47], [SEQ ID NO: 72], [SEQ ID NO: 75]. [SEQ ID NO: 77], and [SEQ ID NO: 89]. The invention further provides conservatively modified variants of these nucleic acids. A "conservatively modified variant" is a variation of nucleic acid that results in a conservative amino acid substitution. In comparison, a "non-conservatively modified variant" is a variation in the nucleic acid sequence that results in a non-conservative amino acid substitution. The means for making these modifications are well known in the art, are described in the examples that follow, and can also be achieved by commercially available equipment and vectors (e.g., New England Biolabs, Inc., Beverly, MA; Clontech , Palo Alto, CA). In addition, the means for evaluating these substitutions (for example, in terms of the effect on binding capacity and introduction to cells) are described in the examples herein.

P1340 / 98 X The means for making this chimeric coat protein, particularly the means for introducing the sequence at the DNA level, are known in the art, illustrated in Figure 2 for a representative chimeric protein, and are described in the examples following. Briefly, the method comprises introducing a sequence (preferably, the sequence of SEQ ID NO: 6 or a conservatively modified variant thereof) into the coat protein sequence. In a preferred embodiment described in the following examples, the introduction is before any finalizing codon or polyadenylation signal to induce a change mutation in the reading frame in the resulting protein, such that the chimeric protein incorporates additional amino acids. In general, this can be achieved by cloning the fiber sequence into a plasmid or some other vector for ease of manipulation of the sequence. Then, the restriction sites that flank the sequence in which the change mutation will be introduced into the reading frame are identified. A synthetic double-stranded oligonucleotide is created from the individual, synthetic, overlapping oligonucleotides, homosense, and antisense oligonucleotides (e.g., from the homosentide and antisense oligonucleotides, P1340 / 98MX respectively TAT GGA GGA TCC AAT AAA GAA TCG TTT GTG TTA TGT TTC AAC GTG TTT ATT TTT C [SEQ ID NO: 9], and AAT TGA AAA ATA AAC ACG TTG AAA CAT AAC ACA AAC GAT TCT TTA TTG GAT CCT CCA [SEQ ID NO: 10], as illustrated in Figure 4) such that the double-stranded oligonucleotide incorporates the restriction sites that they flank the target sequence. The plasmid or other vector is cleaved with the restriction enzymes, and the oligonucleotide sequence having the compatible cohesive ends is ligated to the plasmid or other vector, to replace the wild-type DNA. Another means of sequence directed mutagenesis, in vitro as known to those skilled in the art, and can be achieved (in particular, using PCR), for example, by means of commercially available equipment, can be used to introduce the mutated sequence in the coat protein coding sequence. Once the sequence is introduced into the chimeric protein, the nucleic acid fragment encoding the sequence can be isolated, for example, by PCR amplification using the 5 'and 3' primers. For example, with respect to a chimeric fiber protein, the fragment can be isolated by PCR using the primer TCCC CCCGGG TCTAGA TTA GGA TCC TTC TTG GGC AAT GTA TGA [SEQ ID NO: 11], and the CGT primer GTA TCC ATA TGA CAC AGA [SEQ ID NO: 12], as illustrated in Figure 4. The use of these P1340 / 98 X primers in this manner results in a fragment containing the amplified chimeric fiber that is flanked by the restriction sites (ie, in this case, the Ndel and BamHI sites) that can be used for convenient subcloning of the fragment. Another means can also be used to generate a coat protein. In this way, the change mutation can be introduced into the reading frame in any part of a coding sequence of the coat protein. With respect to SEQ ID NO: 6, for example, that sequence can be placed in the region of the coat protein gene coding for the C-terminus of the protein (i.e., it can be added immediately before the TAA terminator codon) , or can be placed either in the coding region, or as between the codons coding for ALA (ie A), and Gln (ie Q) to produce the aforementioned coding sequence of SEQ ID NO: 8, encoding a chimeric protein comprising the sequence of SEQ ID NO: 5. Similarly, this approach can be used to introduce a change in the reading frame even earlier in the coding sequence, for example, either inserted in or instead of a cover (ie native) protein sequence. In addition, the double-stranded oligonucleotide can P1340 / 98MX also incorporate an additional restriction site that can also be employed in sequence manipulation. For example, the sequence of SEQ ID NO: 6 introduced into the vector comprises a modified BamHI site, ie, the site is "modified" as it adds additional nucleotides on the palindromic recognition sequence. This sequence can also be synthesized to include any other convenient restriction site for DNA manipulations. When incorporated into the coat protein coding sequence, the sequence not only introduces a change mutation of the reading frame, but can also be used to introduce other coding sequences in the coat protein gene. In particular, coding sequences introduced in this manner may comprise codons for lysine, arginine and histidine, or codons for aspartate and glutamate, either alone or in any combination. In addition, a new translation terminator codon can follow these codons for amino acids, allowing a chimeric protein to be produced that only incorporates the given number of additional amino acids into the non-native amino acid sequence. The codons for the amino acids and the transduction terminator codon can be introduced into the new restriction site that induces the change mutation of the P1340 / 98MX reading structure, incorporated into the envelope protein when synthesizing oligonucleotides comprising these flanking sequences by the restriction site as previously described (e.g., comprising the 5 'and 3' BamHI sites), or by other means that are known to those skilled in the art. The size of the DNA used to replace the binding sequence of the native receptor can be restricted, for example, by the prevented folding of the fiber or inappropriate assembly of the penton / fiber base complex. The DNA encoding the amino acid sequences mentioned above (eg, lysine, arginine, histidine, aspartate, glutamate, and the like) is preferred for insertion into the sequence of the fiber gene in which it has been deleted or mutated from another way the sequence binding to the native receptor. In addition, other DNA sequences, such as those encoding amino acids for incorporation in spacing sequence, are used to replace the coding sequences of the native coat protein.

Vector comprising a Chimeric Cover Protein A "vector" according to the invention is a vehicle for gene transfer as that term is understood by those skilled in the art. The four types P1340 / 98MX of vectors encompassed by the invention are plasmids, phages, viruses and liposomes. The plasmids, phages and viruses can be transferred into a cell in their nucleic acid form, and the liposomes can be used to transfer nucleic acids. Therefore, the vectors that can be used for gene transfer are referred to herein as "transfer vectors". Preferably, a vector according to the invention is a virus, especially a virus selected from the group consisting of non-enveloped viruses, i.e., RNA viruses or unwrapped DNA. Also, viruses can be selected from the group consisting of enveloped viruses, i.e., RNA viruses or enveloped DNA. These viruses preferably comprise a coat protein. Desirably, the viral coat protein is one that projects outwardly from the capsid such that it is capable of interacting with the cells. In the case of RNA viruses or enveloped DNA, preferably the envelope protein is actually a lipid envelope glycoprotein (ie, a so-called perforation or peplomer). In particular, preferably a vector is an unwrapped virus (i.e., either an RNA or DNA virus) of the family Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, or Picornaviridae. A preferred non-enveloped virus according to the invention is a virus P1340 / 98MX of the family Hepadnaviridae, especially of the genus Hepadnavirus. A virus of the Parvoviridae family is desirably one of the genus Parvovirus (eg, parvovirus of mammals and birds) or Dependovirus (eg, adeno-associated virus (AAV)). A virus of the family Papovaviridae for example is one of the subfamily Papillomavirinae (for example, the papillomaviruses including, but not limited to, human papillomavirus (HPV) 1-48) or the subfamily Polyomavirinae (for example, including polyomaviruses, but not limited to, JC, SV40 and BK virus). A virus of the Adenoviridae family is desirably one of the genus Mastadenovirus (eg, mammaladenovirus) or Aviadenovirus (eg, poultry adenovirus). A virus of the Picornaviridae family is preferably a hepatitis A virus (HAV), hepatitis B virus (HBV), or a non-A or non-B hepatitis virus. Similarly, a vector may be a virus enveloped in a virus. from the family Herpesviridae or Retroviridae, or it can be a Sindbis virus. A preferred enveloped virus according to the invention is a virus of the Herpesviridae family, especially of the subfamily or genus Alphaherpesvirinae (for example, the virus similar to herpes simplex), Simplex virus (for example, the virus similar to herpes simplex), Varicella virus (for example, P1340 / 98MX viruses similar to varicella and pseudorabies), Betaherpesvirinae (for example, cytomegalovirus), Cytomegalovirus (for example, human cytomegalovirus), Gammaherpesvirinae (for example, the virus associated with lymphocytes), and Lymphocryptovirus (for example, the virus similar to EB). Another preferred enveloped virus is an RNA virus of the Retroviridae family (ie, it is preferably a retrovirus), particularly a virus of the genus or subfamily Oncovirinae, Spumavirinae, Spumavirus, Lentivirinae, and Lentivirus. An RNA virus of the Oncovirinae subfamily is desirably a human T-lymphotropic virus type 1 or 2 (i.e., HTLV-1 or HTLV-2) or bovine leukemia virus (BLV), a poultry leucosis sarcoma virus (for example example, Rous sarcoma virus (RSV), an avmyeloblastosis virus (AMV), poultry erythroblastosis virus (AEV), Rous-associated virus (RAV) -la 50, RAV-O), a mammaltype C virus ( for example, Moloney murine leukemia virus (MuLV), and Harvey's murine sarcoma virus (HaMSV), Abelson's murine leukemia virus (A-MuLV), AKR-MuLV, feline leukemia virus (FeLv) , the virus of simsarcoma, the virus of reticuloendoteliosis (REV), the virus of necrosis of the vessel (SNV), a virus type B (for example, mouse breast tumor virus (MMTV)), or a virus type D (for example, P1340 / 98MX Mason-Pfizer monkey virus (MPMV), "SIDAS" virus). A DNA virus of the Lentivirus virus subfamily is desirably a human immunodeficiency virus type 1 or 2 (i.e., HIV-1 or HIV-2, where HIV-1 is formerly called the virus associated with lymphadenopathy 3 (HTLV-III ) and viruses related to acquired immunodeficiency syndrome (AIDS) (ARV), or another virus related to HIV-1 or HIV-2 that has been identified and associated with AIDS and AIDS-like illnesses. The acronym "HIV" or the terms "AIDS virus" or "human immunodeficiency virus" are used herein to refer to these HIV viruses, and the viruses related and associated with HIV, in a generic manner. In addition, an RNA virus of the Lentivirus subfamily is preferably a Visna / medi virus (e.g., such as from infected sheep), and feline immunodeficiency virus (VLF), bovine lentivirus, simian immunodeficiency virus (SIV), a virus of equine infectious anemia (EIAV, or caprine arthritis-encephalitis virus (CAEV).) An especially preferred vector according to the invention is an adenoviral vector (ie, a viral vector of the Adenoviridae family, optimally of the genus Mastadenovirus Desirably, this vector is an Ad2 or Ad5 vector, although other adenoviral vectors of serotype can be used.

P1340 / 98MX for gene transfer can be natural (that is, competent in replication). Alternatively, the adenoviral vector may comprise genetic material with at least one modification therein, which may render the replication of the virus deficient. Modification to the adenoviral genome may include, but is not limited to, the addition of a DNA segment, rearrangement of a DNA segment, deletion of a DNA segment, replacement of a DNA segment, or introduction of a DNA lesion. . A DNA segment can be as small as one nucleotide and as large as 36 kilobase pairs (ie, the approximate size of the adenoviral genome) or alternatively, it can be ideal at the maximum amount that can be packed into an adenoviral virion (ie say, approximately 38 kb). Preferred modifications to the adenoviral genome include modifications in the El, E2, E3 or E4 region. Similarly, the adenoviral vector can be a co-integrated, i.e., a linkage of the adenoviral sequences, with other sequences, such as another virus or plasmid sequences. In terms of a viral vector (e.g., particularly, an adenoviral vector deficient in replication), this vector may comprise either complete capsids (i.e., which include a viral genome such as an adenoviral genome) or empty capsids (i.e. , in the P1340 / 98MX which is lacking a viral genome, or is degraded, for example, by a physical or chemical means). Along the same lines, since methods are available to transfer viruses, plasmids and phages in the form of their nucleic acid sequences (ie, RNA or DNA), a vector (ie, a transfer vector) it can similarly comprise RNA or DNA, in the absence of any associated protein such as capsid protein, in the absence of any envelope liquid. Similarly, these liposomes affect cell entry by fusing with the membranes of the cell, a transfer vector can comprise liposomes (eg, as they are commercially available, e.g., from Life Technologies, Bethesda, MD ), with the constitutive nucleic acids that code for the coat protein. Thus, according to the invention, while a vector "comprises" a chimeric adenoviral coat protein, a transfer vector "codes for" a chimeric adenoviral coat protein; liposome transfer vectors in particular "code for" in the sense that they may contain nucleic acids that, in fact, code for the protein. A vector according to the invention can comprise additional sequences and mutations, by P1340 / 98MX example, some within the same cover protein. For example, a vector according to the invention preferably further comprises a nucleic acid comprising a passenger gene. A "nucleic acid" is a polynucleotide (DNA or RNA). A "gene" is any nucleic acid gene that codes for a protein or a nascent RNA molecule. A "transient gene" is any gene that is not typically present in a vector, and is subcloned therein (eg, a transfer vector) according to the present invention, and in the introduction into a host cell is accompanied by a discernable change in the intracellular environment (for example, by an increased level of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide or protein, or by an altered rate of production or degradation thereof). A "gene product" is either a still untranslated RNA molecule, transcribed from a given gene or coding sequence (e.g., mRNA or antisense RNA) or the polypeptide chain (i.e., protein or peptide) translated from the mRNA molecule transcribed from the given gene or coding sequence. While the gene comprises coding sequences plus any of the non-coding sequences, a "coding sequence" does not include any non-coding DNA.

P1340 / 98MX encoding (for example, controller). A gene or coding sequence is "recombinant" if the sequence of the bases along the molecule has been altered from the sequence in which nature is typically found to the gene or coding sequence, or if the sequence of Bases are not typically found in nature. In accordance with this invention, a gene or coding sequence can be made synthetically in whole or in part, it can comprise genomic or complementary DNA (cDNA) sequences, and it can be provided in the form of either DNA or RNA. The non-coding sequences or regulatory sequences include promoter sequences. A "promoter" is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. "Enhancers" are cis-acting elements of DNA that stimulate or inhibit the transcription of adjacent genes. An intensifier that inhibits transcription is also called a "silencer." Enhancers require DNA binding sites for the specific DNA binding proteins of the sequence found only in the promoter (which are also called "promoter elements") in which the enhancers can function in any orientation, and over distances of up to several kilobase pairs, even from a position in the P1340 / 98MX 3 'direction of a transcribed region. According to the invention, a coding sequence is "operably linked" to a promoter (eg, when both the coding sequence and the promoter constitute a passenger gene) when the promoter is capable of directing the transcription of this coding sequence. . Accordingly, a "passenger gene" can be any gene, and desirably is any of a therapeutic gene or an indicator gene. Preferably, a passenger gene is capable of being expressed in a cell in which the vector is internalized. For example, the passenger gene may comprise a reporter gene, or a nucleic acid sequence that encodes a protein that can be detected in some way in a cell. The passenger gene can also comprise a therapeutic gene, for example, a therapeutic gene that exerts its effect at the RNA or protein level. For example, a protein encoded by a transferred therapeutic gene can be extended in the treatment of a hereditary disease, such as, for example, the cDNA regulating the transmembrane conductance of cystic fibrosis for the treatment of cystic fibrosis. The protein encoded for the therapeutic gene can exert its therapeutic effect by resulting in cell death. For example, expression of the gene itself can lead to cell death, as with P1340 / 98MX gene expression of diphtheria toxin A, or gene expression can return to cells selectively sensitive to the killing action of certain drugs, for example, the expression of the HSV thymidine kinase gene returns to cells sensitive to antiviral compounds including acyclovir, ganciclovir and FIAU (1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-iodouracil). In addition, the therapeutic gene can exert its effect at the RNA level, for example, by coding an antisense message or ribosome, a protein that affects introns removal or 3 'processing (eg, polyadenylation), or it can code for a protein that acts by affecting the level of expression of another gene within the cell (ie, where expression of the gene that includes all steps from the initiation of transcription through the production of a processed protein is widely considered), perhaps, among other things, by mediating an altered speed of mRNA combination, an alteration of mRNA transport, and / or a change in post-transcriptional regulation. Accordingly, the use of the term "therapeutic gene" is proposed to mark these and any other modalities of what is most commonly referred to as gene therapy and is known to those skilled in the art. Similarly, recombinant adenovirus can be used for gene therapy or for P1340 / 98MX study the effects of gene expression in the given cell or tissue in vitro or in vivo. The recombinant adenovirus comprising a chimeric coat protein such as a fiber protein and the recombinant adenovirus additionally comprising a passenger gene or genes capable of being expressed in a particular cell, can be generated by the use of a transfer vector, preferably a viral or plasmid transfer vector, according to the present invention. This transfer vector preferably comprises a gene sequence of chimeric adenoviral coat protein as previously described. The chimeric coat protein gene sequence comprises a non-native sequence in place of the native sequence, which has been deleted, or in addition to the native sequence. A recombinant chimeric coat protein gene sequence (such as a fiber gene sequence) can be moved from an adenoviral transfer vector in the baculovirus or a prokaryotic or eukaryotic expression vector suitable for the expression and evaluation of the specificity and avidity of the receptor or protein, potential and trimerization, attachment to the penton base, and other biochemical characteristics. Accordingly, the present invention also P1340 / 98MX provides baculoviral and prokaryotic and eukaryotic expression vectors, recombinants, which comprise a chimeric adenoviral envelope protein gene sequence (preferably a fiber gene sequence), which are also "transfer vectors" as defined herein. The gene sequence of chimeric coat protein, (e.g., fiber gene sequence) includes a non-native sequence of, or in place of, the native amino acid sequence, and which allows the resulting chimeric protein to be coated (for example). example, the fiber protein) binds to a different binding site of a binding site linked by the native sequence. By moving the chimeric gene from an adenoviral vector to the baculovirus or a prokaryotic or eukaryotic expression vector, high protein expression (approximately 5-50% of the total protein which is the chimeric fiber) can be achieved. A vector according to the invention may further comprise, either within, or instead of, or outside of the coding sequence of the additional coat protein sequences that impact the ability of a coat protein such as fiber protein. to trimerize, or comprise a protease recognition sequence. A sequence that impacts the ability to trimerize is one or more sequences that allow the P1340 / 98 X trimerization of a chimeric coat protein that is a fiber protein. A sequence comprising a protease knowledge sequence is a sequence that can be cleaved by a protease, thereby affecting the removal of the chimeric coat protein (or a portion thereof) and the binding of the recombinant adenovirus to a cell by means of another cover protein. When employed with a coat protein that is a fiber protein, the protease recognition site preferably does not affect fiber trimerization or specificity of the fiber protein receptor. For example, in one embodiment of the present invention, preferably, the fiber protein, or a portion thereof, is deleted by means of a protease recognition sequence, and then the new cell surface binding site it is incorporated in either the penton base protein or the exon coating, preferably with the use of a spacer sequence as previously described. In terms of the production of vectors and transfer vectors according to the invention, the transfer vectors are constructed using normal genetic and molecular techniques as are known to those skilled in the art. Vectors (for example, virions or virus particles) are produced using P1340 / 98MX viral vectors. For example, a viral vector comprising a chimeric coat protein according to the invention can be constructed or provided to a cell that does not comprise any complementary sequence E4: (1) a linear vector comprising the chimeric fiber and the natural E4 gene , and (2) a linear vector that is E4 ', as illustrated in Figure 3. As described in the examples that follow, this methodology results in recombination between the sequences, the generation of a vector comprising a portion of the initial E4 'vector and a portion of the E4 + vector, particularly the region comprising the chimeric fiber sequences. Similarly, particles containing the fiber chimera are produced in normal cell lines, for example, those commonly used for adenoviral vectors. After the production and purification, the particles in which the fiber is to be suppressed become without fiber through the digestion of the particles with a specific protease of the sequence, which cleaves the fiber proteins and releases them from the fibers. Viral particles to generate the particles in fiber. For example, thrombin is recognized and cleaved to known amino acid sequences that can be incorporated into the vector (Stenflo et al., J. Biol. Chem., 257, 12280-12290 (1982)). Similarly, the suppression of P1340 / 98MX mutants lacking the fiber gene can be employed in the construction of the vector, for example, H2dl802, H2dll807, and Hdll021 (Falgout et al., J. Virol., 62, 622-625 (1988). fiber has been known to be stable and capable of cell attachment and infection (Falgout et al., supra) .These resulting particles can then be objectified to specific tissues via the base of penton and other coat protein, preferably another coat protein. such that it comprises one or more non-native amino acid sequences according to the invention Alternatively, the recombinant adenovirus comprising the chimeric fiber protein having additional modifications may be produced by the removal of the native leaving region, which comprises the domains of trimerization and receptor binding of the fiber protein and its replacement with a non-native trimerization domain (Peteranderl et al., Biochesmit ry, 31, 12272-12276 (1992)) and a non-native amino acid sequence according to the invention. A recombinant adenovirus comprising a chimeric fiber protein can also be produced by mutation of points in the leaving region and isolation of clones that are capable of trimerization. In any case, and also with respect to the removal of replacement of the sequence of P1340 / 98MX specific receptor binding, native, as described above, new protein binding domains can be added in the C-terminus of the fiber protein or in the exposed loops of the fiber protein by inserting one or more copies of the nucleic acid sequence encoding the non-native sequence of amino acids at the appropriate position. Preferably, this fiber protein is capable of trimerizing, so that it is capable of binding to the penton base protein. The method described above for generating the chimeric fiber protein can also be used to make other chimeric coat proteins, for example chimeric exon or penton proteins.

Illustrative Uses The present invention provides a chimeric protein that is capable of binding to cells and mediating entry into cells with high efficiency, as well as vectors and transfer vectors comprising the same. The chimeric coat protein itself has multiple uses, for example, as a tool for in vitro studies of adenoviruses that bind to cells (for example, with Scatchard analysis as previously shown by Wickham et al. (1993), supra). ), to block the binding of the adenovirus to the receptor in vitro (e.g.

P1340 / 98MX use antibodies, peptides and enzymes, as described in the examples), and to protect against adenoviral infection in vivo by competing for binding to the binding site by which the adenovirus enters the cell. A vector comprising a chimeric coat protein can also be used in the generation of strains and as a means to elaborate new vectors. For example, the non-native sequence of amino acids can be linked to nucleic acids, and can be introduced intracellularly as a means to generate new vectors via recombination. Similarly, a vector can be used in gene therapy. For example, a vector of the present invention can be used to treat any of a number of diseases by distributing to target cells the corrective DNA, i.e., the DNA encoding a function that is either absent or damaged, or a discrete killer agent, for example, a DNA encoding a cytotoxin which, for example, is active only intracellularly. Diseases that are candidates for this treatment include, for example, cancer, for example, melanoma, glioma or lung cancers; genetic disorders, for example, cystic fibrosis, hemophilia or muscular dystrophy, pathogenic infections, for example human immunodeficiency virus, tuberculosis P1340 / 98MX or hepatitis; heart disease, for example, to prevent restenosis following angioplasty or promote angiogenesis for reperfusion of necrotic tissue; and autoimmune disorders, for example, Crohn's disease, colitis or rheumatoid arthritis. In particular, gene therapy can be carried out in the treatment of diseases, disorders, conditions associated with different tissues that ostensibly lack high levels of the receptor to which the natural adenovirus fiber protein binds, and thus for the which standard treatments mediated by adenovirus to gene therapy are less optimal (for example, for the distribution to cells of monocytes / macrophages, fibroblasts, neurons, smooth muscle, and epithelial cells). The tissues comprised of these cells (and diseases, disorders, or conditions associated therewith) include, but are not limited to: endothelial (eg, angiogenesis, restenosis, inflammation, and tumors); neuronal tissue (e.g., tumor and Alzheimer's disease); epithelium (for example, disorders of the skin, cornea, intestines and lung); hematopoietic cells (e.g., human immunodeficiency virus (HIV-1, HIV-2), cancers and anemias); smooth muscle (eg, restenosis); and fibroblasts (e.g., inflammation).

P1340 / 98MX In addition, instead of transferring a therapeutic gene, a reporter gene or some type of marker gene can be transferred instead using the vectors (particularly the adenoviral vectors) of the invention. Marker genes and reporter genes are of use, for example, in cell differentiation and cell death studies, as well as potentially for diagnostic purposes. In addition, a normal reporter gene such as a β-galactosidase reporter gene, a gene encoding for the green fluorescent protein (GFP), or a β-glucuronidase gene can be used in vivo, for example, as a test medium in a live host, or for example, as a means of removing targeted cells (see, for example, Minden et al., Biotechniques, 20, 122-129 (1996); Youvan, Science, 268, 264 (1995); U.S. Patent No. 5,432,081; Deonarain et al., Br. J. Cancer, 70, 786-794 (1994)). Similarly, it may be desirable to transfer a gene to use a host essentially as a means of in vivo production of a particular protein. Along these lines, transgenic animals have been used, for example for the production of recombinant polypeptides in the milk of transgenic bovine species (for example, International PCT Application).

P1340 / 98MX WO 93/25567). Other "non-therapeutic" reasons for gene transfer are the study of human diseases using an animal model (eg, the use of transgenic mice and other transgenic animals including deletions of p53 tumor suppressor genes for tumorigenic studies, the use of a model transgenic to impart glucose tolerance and models of Alzheimer's amyloid precursor protein, human, for the study of glucose metabolism and the pathogenesis of Alzheimer's disease, respectively, etc.). These illustrative uses mentioned above are not very broad, and it is proposed that the present invention encompass these additional uses that come, but are not explicitly cited, in the description herein. Similarly, there are numerous advantages associated with the use of various aspects of the present invention. For example, the use of a universal selection vector of targets according to the invention is advantageous considering that: (1) the vector can potentially be used for all cells and tissues; (2) only one vector is required for use in all cell lines, there is no need to co-transfect an independent vector; (3) vector capable of effecting gene distribution with an efficiency that increases over that observed for vectors that comprise fiber protein P1340 / 98MX from natural; (4) the vector, different from previous vectors, does not target specific cells, but instead increases transduction sufficiency since it seems to be a global way; (5) vector capable of mediating gene transfer when employed at a reduced dose (i.e., multiplicity of infection (MOI)) compared to the vector comprising the natural fiber protein, and thus likely reduces the disadvantages related to the doses that accompany the currently available adenoviral vectors; and (6) the vector can be propagated and maintained using currently available cell lines. The ability of a universal targeting vector such as an adenovirus vector of universal targeting to potentially bind to and enter all or most of the tissues has several advantages. These advantages include increased efficiency or gene distribution to multiple tissues, and the availability of an individual vector capable of distributing genes to all tissues, and the simplified production of the components necessary for gene distribution. In addition, this universal targeting vector comprises a potential to distribute exogenous DNA in cells by "carrying on the shoulders" the DNA in the vector by means of a protein / DNA interaction. P1340 / 98MX The additional potential advantages of this universal target selection vector include a substantially increased efficiency of distribution (eg, increased by 10 to 100 times) in cells expressing low levels of the fiber receptor to which the natural fiber protein binds, as well as increased efficiency in cells or tissues that express the fiber receptor to which the fiber binds natural. In addition, the reduced doses at which the vectors are employed should result in a decrease in the inflammation associated with adenovirus, the humoral response to adenovirus, and the cytotoxic T lymphocyte response to adenoviruses. In addition, the vector is advantageous since it can be isolated and purified in any conventional medium. These changes in the vector are made at the genome level, there are no heavy modifications and expensive post-production required, as is associated with other vectors (see, for example, Cotten et al., Supra).; Wagner et al., Supra). Similarly, cell lines that express the special receptor are not required. A UTV vector can be propagated to similar titers as a natural vector that lacks the fiber modification.

P1340 / 98MX Means of Administration The vectors and transfer vectors of the present invention can be used to contact the cells either in vitro or in vivo. According to the invention, "contacting" comprises any means by which a vector is introduced intracellularly; the method is not dependent on any particular means of introduction and should not be limited in this way. The means of introduction are well known to those skilled in the art, and are also exemplified herein. Accordingly, introduction can be effected, for example, either in vitro (for example, in an ex vivo type method of gene therapy or in tissue culture studies) or in vivo by electroporation, transformation, transduction, conjugation or pairing triparental, (co-) transfection, (co-) infection, membrane fusion with cationic lipids, high-speed bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection in individual cells and the like. Similarly, the vectors can be introduced by means of cationic lipids, for example, liposomes. These liposomes are commercially available (e.g., Lipofectin R, Lipofectamine ™, and the like, supplied by P1340 / 98MX Life Technologies, Gibco BRL, Gaithersburg, MD). In addition, in liposomes having increased transfer capacity and / or reduced toxicity in vivo (see, for example, PCT Patent Application WO 95/21259) can be employed in the present invention. Other methods are also available and are known to those skilled in the art. According to the invention, a "host" (and thus a "cell" of a host) encompasses any host in which a vector of the invention can be introduced, and thus encompasses an animal, including, but not limited to, an amphibian, bird, fish, insect, reptile or mammal. Optimally, a host is a mammal, for example, a rodent, primate (such as chimpanzee, monkey, ape, gorilla, orangutan or gibbon), feline, canine, ungulate (such as ruminant or pig), as well as in particular, human . In view of the fact that a universal selection vector of targets ostensibly enters all cells, a cell can be any cell in which this vector can enter. In particular, a universal targeting vector can be employed for gene transfer to a cell expressing low or undetectable levels of the fiber receptor, including, but not limited to an endothelial, smooth muscle cell, Neuronal, hematopoietic, or fibroblast P1340 / 98MX. One skilled in the art will appreciate that suitable methods are available for the administration of a vector (particularly an adenoviral vector) of the present invention to an animal for gene therapy purposes (see, eg, Rosenfeld et al., Science, 252, 431 -434 (1991); Jeffe, et al., Clin. Res., 39 (2), 302A (1991), Rosenfeld et al., Clin. Res. 39 (2), 311A (1991); Berkner, BioTechniques, 6, 616-629 (1988)), chemotherapy and vaccination, and although more than one route can be used for administration, a particular route may provide a more immediate and more effective reaction than another route. Pharmaceutically acceptable excipients are also well known to those skilled in the art, and are readily available. The choice of excipient will be determined in part by the particular method used to administer the recombinant vector. Accordingly, there is a wide variety of formulations suitable for use in the context of the present invention. The following methods and excipients are exemplary only and are not limiting in this way. Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or P1340 / 98MX orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, such as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. The tablet forms may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, sodium cross carmellose, talcum, magnesium stearate, stearic acid, and others. excipients, colorants, diluents, buffering agents, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. The tablet forms may comprise the active ingredient in a flavor, usually sucrose or gum arabic, or tragacanth, as well as tablets comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and gum arabic, emulsions, gels and the like containing, in addition to the active ingredients, these excipients as are known in the art. A vector or recombinant vector of the present invention, alone or in combination with other suitable components, can be made in an aerosol formulation to be administered via inhalation. .These aerosol formulations can be placed in acceptable pressurized propellants such as dichlorodifluoromethane, propane, P1340 / 98MX nitrogen and the like. They can also be formulated as pharmaceuticals for non-pressurized preparations such as in a nebulizer or an atomizer. Formulations suitable for parenteral administration include solutions of weak, aqueous and non-aqueous, isotonic injections, which may contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the proposed recipient, and sterile aqueous suspensions and non-aqueous suspensions. aqueous containing dispersing agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations may be presented in sealed unit dose or multiple dose containers, such as ampoules and flasks, and may be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid excipient, eg, water, for injections, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Additionally, a vector or transfer vector of the present invention can be made in suppositories by mixing with a variety of bases such as emulsifying bases or water soluble bases.

P1340 / 98MX Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, carriers as are known in the art to be appropriate. The dose administered to an animal, particularly in humans, in the context of the present invention will vary with the gene of interest, the composition employed, the method of administration, and the particular site and organism being treated. However, the dose should be sufficient to effect a therapeutic response. As previously indicated, a vector or a transfer vector of the present invention also has in vitro utility. This vector can be used as a search tool in the study of cell attachment and adenoviral infection and in a method of assaying the interaction of the binding site-ligand. Similarly, the recombinant coat protein comprising a non-native amino acid sequence in addition to or instead of a native receptor binding sequence can be used in receptor-ligand assays and as in vitro or in vivo addition proteins. , for example. The following examples further illustrate P1340 / 98MX the present invention, and of course, should not be considered in any way limiting its scope.

Example 1 This example describes an investigation of the levels of the adenovirus receptor in different cells, as determined by the ability of the natural adenovirus to bind to the cells. For these studies, the ability of the adenovirus comprising the natural fiber to bind to cells derived from various tissues was assessed. The adenovirus particles of an Ad5 strain were labeled with [3 H] -thymidine as previously described (see, for example Wickham et al., Cell, 73, 309-319 (1993)). The subsaturation levels of the thymidine-labeled adenovirus were labeled at 200 μl to 10 pre-incubated cells for approximately 30 to 60 minutes with or without 20 μg / ml of soluble fiber protein. The cells were incubated with the virus for 1 hour at 4 ° C and then washed 3 times with cold phosphate buffered saline (PBS). The beads associated with the remaining cells were eluted in a scintillation counter. The specific binding was measured by subtracting counts associated with cells (ie, counts per minute (cpm)) in the presence of fiber from the cell-associated accounts in the absence of P1340 / 98 X fiber. At the junction in the presence of fiber was never more than 2% of the total entry of radioactive virus particles. The results were obtained, on average, in triplicate missions. As illustrated in Figure 1, a substantial number of cells derived from different tissues expressed little or no fiber receptor, as indicated by a relative inability of the native adenovirus to bind to these cells. Cells of epithelial origin (ie, "recipient-plus" cells that include Chang, HeLa, and A549 cells) bound high levels of adenovirus. In comparison, non-epithelial cells (i.e., "minus-receptor" cells such as monocyte / macrophage cells, fibroblasts, neuronal, smooth muscle, and epithelial cells) exhibited approximately 10-fold or fewer reductions in virus binding in comparison to cells similar to epithelial cells. These results confirm the previously unrecognized relative inability of the adenovirus to bind to the non-epithelial cells, of receptor-minus, and therefore to enter them, in comparison with the epithelial cells of receptor-plus. Presumably, this inability of life to the low representation of the natural adenoviral fiber protein receptors in these cells.

P1340 / 98MX Example 2 This example describes the construction of an adenoviral vector comprising a chimeric coating protein, particularly a chimeric adenoviral fiber protein. To overcome the transduction limitation imposed by the presence of only a limited number of fiber receptors in clinically relevant tissues such as non-epithelial tissue, a modified adenovirus vector was constructed as depicted in Figures 2A and 2B to derive a vector referred to herein as a "universal transfer vector", or UTV. In particular, a change mutation was created in the reading frame in a gene that codes for a coated adenoviral protein, in this case, in the fiber gene. In the natural adenovirus, the unmodified fiber gene contains a pooled translational finalizing signal (TAA) and the transcriptional polyadenylation signal (AATAAA). The polyadenylation signal directs the addition of a polyA prolongation at the 3 'end of the transcript. The polyA extension typically comprises anywhere from about 20 to about 200 nucleotides. After transcription and exit from the nucleus, the TAA finalizing signal directs the termination P1340 / 98MX of the translation with ribosome. In comparison, the modified fiber gene of a UTV vector lacks a "finalizing" translational signal in the reading structure. After normal transcription and the addition of the polyA extension in the ARMm, in the absence of the finalizing codon, the ribosome continues the translation of the transcript in the polyA region. In view of the codon AAA encoding the amino acid lysine, the resultant chimeric fiber gene translation product produced by a UTV contains an addition of a polylysine residue sequence in the C-terminus, ie, Lys Lys Lys Lys Lys Lys Lys Lys [SEQ ID NO: 1]. It is possible for a cellular process to act to limit the length of the polylysine sequence, since the polylysine residues typically from about 3 to about 30 residues in the chimeric fiber protein. Whatever the case may be, however, the polylysine protein modification, as well as the additional modifications described herein, allow the UTV to efficiently attack cells lacking high levels of the receptor for the fiber, adenoviral, natural protein (s). say, receptor-less cells). In terms of the construction and characterization of the vector, the normal molecular and genetic techniques, P1340 / 98MX such as generation of strains, plasmids, and viruses, gel electrophoresis, DNA manipulations including plasmid isolation, DNA cloning and sequencing, Western blotting assays, and the like, were performed as known to those skilled in the art, and as described in detail in standard laboratory manuals (eg, Molecular Cloning: A Laboratoy Manual, 2nd ed. (Cold Spring Harbor, NY, 1992); Ausubel et al., Current Protocols in Molecular Biology, (1987)). Restriction enzymes and other enzymes used for molecular manipulations were purchased from commercial sources, for example, Boehringer Mannheim, Inc., Indianapolis, Indiana, New England Biolabs, Beverly, Massachusetts; Bethesda Research Laboratories, Bethesda, Maryland), and were used in accordance with the manufacturer's recommendations. Cells used for the experiments (e.g., 293 kidney, embryonic, human, transformed cell lines (i.e., CRL 1573 cells) and other cells listed by the American Type Culture Collection) were cultured and maintained using reagents Normal, medium and technical sterile cultures, as previously described (Erzerum et al., Nucleic Acids Research, 21, 1607-1612 (1993)). Therefore, the change mutation in the P1340 / 98MX 'reading structure of the fiber terminator codon was created by introducing a modified BamHI site (i.e., GGATCCAA [SEQ ID NO: 6]) into an adenoviral transfer vector. This was done as illustrated in Figure 3 when starting with the transfer plasmid pAd NS 83-100 (which is also known as pl93NS 83-100 or pNS 83-100). PAd NS 83-100 was constructed by cloning the Ndel fragment of Ad5 to the SalI fragment, which extends over the unitary region of the Ad5 genome map 83-100 contag the fiber gene, in the plasmid pNEB193 (New England Biolabs , Beverly, MA). The Ndel-Munl fragment from pAd NS 83-100 was replaced with a synthetic oligonucleotide comprising a BamHI site, which was flanked by a 5 'Ndel site and a 3' Muñi site to facilitate cloning. The double-stranded synthetic oligonucleotide fragment was created from the superposition of the single-stranded, synthetic strand homosense and antisense oligonucleotides, ie, respectively, the antisense primer TGA GGA TGA AAT AAA GAA TCG TTT GTG TTA TGT TTC AAC GTG TTT ATT TTT C [SEQ ID NO: 9], and the antisense primer AAT TGA AAA ATA AAC ACG TTG AAA CAT AAC ACA AAC GAT TCT TTA TTG GAT CCT CCA [SEQ ID NO: 10], as illustrated in the Figures 4A and 4B, respectively. The ends of the superimposed oligomers were made to have compatible protrusions P1340 / 98MX for direct cloning at the Ndel and Muñí sites. The resulting transfer plasmid, pAd NS 83-100 (F ~) (which is also known as pl93NS (? F) or pNS (? F)), lacks the first 50 base pairs of the coding sequence for the gene fiber (that is, it is "fiber-less"). The vector also contains the complete E4 coding sequence of adenovirus. The plasmid retains the AATAAA polyadenylation signal included in the synthetic Ndel / Munl oligonucleotide, and also incorporates the new BamHI restriction site. The mutated fiber gene was incorporated into the fiber-less plasmid pAd NS 83-100 using synthetic, antisense and antisense oligonucleotide primers to amplify the fiber end with the use of the polymerase chain reaction (PCR) while that a modified BamHI site is incorporated after the last codon of the fiber gene to create the mutant fiber gene. This modified, incorporated BamHI site also serves to encode amino acids, glycine and serine, resulting in a chimeric nucleic acid sequence of GGA TCC AAT AAA GAA TCG TTT GTG TTA TGT [SEQ ID NO: 7]. The modified fiber gene encodes in this manner for an extension to the resulting fiber chimeric protein Gly Ser Asn Lys Glu Ser Phe Val Leu Lys Lys Lys [SEQ ID NO: 4], wherein the length of the polylysine sequence may vary . The P1340 / 98MX synthetic oligonucleotides used for the first amplification were the primer TCCC CCCGGG TCTAGA TTA GGA TCC TTC TTG GGC AAT GTA TGA [SEQ ID NO: 11], and the primer CGT GTA TCC ATA TGA CAC AGA [SEQ ID NO: 12] , as illustrated in Figures 4C and 4D, respectively. The amplified gene product was then cut with the restriction enzymes Ndel and BamHI, and cloned into the Ndel / BamHI sites of the fiber plasmid-minus pAd NS 83-100 to create the transfer vector pAd NS 83-100 UTV (which also it is known as pl93NS (F5 *), pl93 (F5 *), or pNS (F5 *)). The complete adenovirus Ndel to Sali sequence from pAd NS 83-100 UTV was cloned into the fiber plasmid-minus pAd BS 59-100 to create pAd BS 59-100 UTV (which is also known as pl93 NS (F5 *), pl93 (F5 *), or pNS (F5 *). The adenovirus vector UTV was created through homologous recombination in CELLS 293. Specifically, the transfer vector E4 * pAd BS 59-100 UTV was linearized with SalI, and transfected into 293 cells that were previously infected with the adenovirus vector, A2F.The A2F vector was derived from a GV10 vector.The GV10 vector on Ad5 base contains the lacZ gene under the control of the Rous sarcoma virus promoter ( that is, it comprises RSV lacZ.) The insertion of the indicator in GV10 gene is done within the El region (ie, the vector is El '). The vector GV10 also contains a deletion of the P1340 / 98MX region E3, but it is E4 '. In comparison with GV10 (ie, RSV lacZ 'E3' E4 '), A2F additionally comprises a deletion of the essential E4 adenovirus genes, but it is E3' (ie RSV lacZ The 'E3' E4 '). Cells 293 contain a complementary sequence El, but do not contain a complementary sequence E4. The lack of a complementary sequence E4 prevents the replication of the A2F vector of E4 in the 293 cell line. However, in the co-introduction of the A2F virus and pAd BS 59-100 UTV in 293 cells, the homologous recombination takes place between the recombinant vector UTV and the adenoviral genome A2F, producing an E3 'E4' adenovirus genome comprising a chimeric fiber protein, which is capable of 293 cell replication. This resulting, particular UTV vector was designated GV10 UTV. The GV10 UTV vector was isolated using normal plate isolation techniques with 293 cells. After three successive rounds of plaque purification, the GV10 UTV vector contained the fiber mutation and was free of any contamination by the E4 'A2F vector . The presence of the chimeric fiber sequences in the vector GV10 UTV was confirmed by sequencing the fiber mRNA using the reverse transcriptase-polymerase chain reaction (RT-PCR), which validated the presence of a P1340 / 98MX prolongation of polyadenine in the chimeric fiber mRNA. Similarly, the production of the chimeric fiber protein by the vector was continued by Western blotting. To accomplish this, 293 cells were infected at a multiplicity of infection (MOI) of 5 with either GV10 comprising the natural fiber adenoviral protein or GV10 UTV comprising the chimeric fiber protein. Two days post-infection, the cells were washed and lysed in PBS for three freeze-thaw cycles. The lysates were cleaned by centrifugation and loaded on a 10% sodium dodecyl sulfate gel / polyacrylamide. After electrophoresis, the proteins were transferred into nitrocellulose and detected by chemiluminescence using a polyclonal antibody to the fiber. The Western blot is represented in Figure 5. As can be seen from this figure, the migration of the proteins indicates that the UTV chimeric fiber is from about 1.5 up to protein 2.0 kilodaltons larger than the WT fiber protein from 62 kilodalton, not modified. These results confirm that the method identified herein can be used to introduce modifications in the fiber protein to produce a chimeric fiber protein. Similar techniques can be used to introduce modifications P1340 / 98 X in the exon or penton proteins, or to introduce similar modifications (for example, the addition of an amino acid sequence comprised of arginine, lysine and / or histidine, comprised of aspartate and / or glutamate, or the addition of any of these sequences in a coding region of the coat proteins.

EXAMPLE 3 This example describes the cell attachment of an adenoviral vector comprising a chimeric coat protein such as a chimeric fiber protein as compared to a natural adenoviral vector, either in the presence or absence of natural fiber protein, per soluble . For these experiments, the cells identified in Example 1 were used, to which adenoviruses bind with either high efficiency (ie, receptor-plus cells) or low efficiency (ie, receptor-minus cells). The epithelial cell line A549 was used as representative of the receptor-plus cells, and the fibroblast cell line HS 68 was used as representative of the receptor-minus cells. The confluent monolayers of either A549 or HS68 cells were pre-incubated at 4 ° C with soluble fiber protein concentrations ranging from 0 to about 10 μg / ml. The vector GV10 UTV P1340 / 98 X comprising the chimeric fiber protein (UTV) or the vector GV10 comprising the natural fiber protein (WT) were labeled with tritiated thymidine as described in Example 1. Approximately 20,000 cpm of GV10 UTV labeled with [ 3H] -thymidine or GV10 vector was then incubated with the cells for approximately 2 hours at 4 ° C. The cells were washed three times with cold PBS, and the cpm associated with the cell was determined by scintillation counting. The results were obtained as the average of duplicate measurements and are presented in Figures 6A and 6B for cell lines A549 and HS 68, respectively. As can be seen in Figures 6A and 6B, the chimeric fiber protein of the vector GV10 UTV was able to bind to both the receptor-plus cells (Figure 6A) and the receptor-minus cells (Figure 6B) with high efficiency. In comparison, the GV10 vector comprising the natural fiber was more effective in binding to the receptor-plus cells. In particular, radiolabelled UTV GV10 bound to cells expressing detectable levels of the fiber receptor (i.e., alveolar epithelial cells A549) approximately 2 to 2.5 times better than GV10. While the entire binding of the GV10 vector was inhibited by converting the recombinant fiber protein, only about 40% of the GV10 UTV vector is inhibited by the addition of the competing fiber. No union was observed P1340 / 98MX detectable GV10 vector comprising natural adenoviral fiber to HS 68 human foreskin fibroblast cells lacking fiber receptor. In comparison, the GV10 UTV receptor efficiently bound to the HS 68 cells, and the addition of the competition fiber protein did not affect the binding. These results confirm that binding of the GV10 UTV vector comprising a chimeric coat protein (i.e., a chimeric fiber protein) does not occur via the natural adenoviral fiber receptor, instead it occurs via an unrecognized fiber receptor to date. . In addition, the results confirm that incorporation and chimeric coat protein such as a chimeric coat protein in an adenoviral vector results in an improved adenoviral vector. Specifically, the modification comprised by the vector GV10 UTV allows to overcome the relative inability mentioned above of the natural adenovirus to bind to the receptor-minus cells, in particular, non-epithelial cells, and also allows the modified vector to bind to the recipient cells. more with increased efficiency.

Example 4 This example describes an investigation of the P1340 / 98MX capacity of several soluble factors, and inhibitors of these soluble factors, to block the binding of the adenovirus comprising chimeric fiber protein to the fibers of HS-receptor-minus fibroblasts. For these experiments, the inhibition of GV10 UTV binding was evaluated by several negatively charged molecules including salmon sperm DNA, mucin, chondroitin sulfate and heparin. Chondroitin sulfate and heparin are negatively charged molecules that obtain their charge from the sulfate groups. The mucin is negatively charged due to the presence of the sialic acid moieties, and the DNA is negatively charged due to its incorporation of phosphate moieties. Approximately 20,000 cpm of UTV in 250 μl of binding buffer (i.e., Dulbecco's Modified Eagle Medium (D-MEM) was incubated at room temperature for 30 minutes with concentrations of negatively charged molecules ranging from about 1 x 10 ~ 3 to about 1 x 10 μg / ml After incubation, the mixtures were chilled on ice, and then added to pre-cooled HS 68 cells placed in 24-well plates.The cells were incubated for about 1 hour, and then the cells were washed three times with PBS The cpm associated with cells were determined by scintillation counting, and were reported P1340 / 98MX as the average of reported measurements. As indicated in Figure 7, while the presence of the natural competent fiber protein had no effect on the binding of a GV10 UTV vector (i.e., comprising chimeric fiber) to the HS 68 cells, the charged competent molecules negatively they were able to block the binding of GV10 UTV. All four molecules were able to inhibit the binding of GV10 UTV to HS 68 cells, although heparin and DNA were more effective. These molecules have no significant effect on the binding of a GV10 vector (ie, comprising the natural fiber) to cells expressing high levels of the fiber receptor (ie, A549 cells, data not shown). These results confirm that the negatively charged molecules are capable of blocking the binding of the GV10 UTV vector mediated by the chimeric fiber protein. This inhibition is presumably due to the binding of the negatively charged molecules to the positively charged polylysine residues present in the GV10 UTV fiber. Accordingly, the impact of the enzymes that cleave these negatively charged molecules on binding to the cells of the GV10 UTV vector was assessed. HS 68 cells were placed in 24-well plates, and pre-incubated with the heparinase dilutions P1340 / 98MX (Sigma, St. Louis, MO), chondroitinase (Sigma), and sialidase (Boehringer Mannheim, Inc.) ranging from about 0.0001 to 1 for 45 to 37 ° C, followed by 15 minutes at 4 ° C. While chondroitinase is simple chondroitin sulfate, heparinase cleaves heparin and heparin sulfate, and sialidase clears sialic acid. The initial concentrations for the dilutions were as follows: heparinase, 25 U / ml (U = 0.1 μmol / hour, pH = 7.5, 25 ° C); chondroitinase, 2.5 U / ml (U = 1.0 μmol / minute, pH = 8.0, 37 ° C); and sialidase 0.25 U / ml (U = 1.0 μmol / minute, pH = 5.5, 37 ° C). After incubation, the cells were washed three times with cold PBS, then incubated with 20,000 cpm of the labeled GV10 UTV vector for approximately 1 hour at 4 ° C. The cells were then washed with cold PBS and the cpm associated with cells were determined by scintillation counting. The results were reported as the average of the duplicate measurements. As illustrated in Figure 8, pretreatment of HS 68 cells with enzymes to remove negatively charged molecules from the cell surface confirms that the GV10 UTV vector comprising the chimeric fiber protein interacts with the charged sites relatively on the cell surface . In particular, heparinase and sialidase were both able to reduce the binding of GV10 P1340 / 98MX UTV, although heparinase was more effective than sialidase in HS 68 cells. Thus, these results confirm that a vector comprising the chimeric fiber protein (e.g., a GV10 UTV vector), different from adenovirus natural, interacts in a new way with negatively charged molecules on the cell surface to effect entry into the cell. These results further demonstrate that a vector comprising negatively charged residues (e.g., aspartate and glutamate) instead of positively charged molecules (e.g., lysine) can be used in a similar manner to bind to, and effect entry into, the cell via the positively charged molecules present on the cell surface.

Example 5 This example evaluates the gene distribution to different cell types mediated by an adenoviral vector comprising the chimeric coat protein as a chimeric fiber protein (eg, GV10 UTV) as compared to the adenovirus-mediated gene distribution comprising the natural cover protein such as fiber protein (eg, GV10). For these experiments, the relative levels of the lacZ gene distribution was a vector containing the P1340 / 98MX natural fiber protein (ie, GV10) compared to the vector containing the chimeric fiber protein (ie, GV10 UTV) were compared in epithelial-like cells (ie HeLa cells, A549, HepG2 and H700 T), smooth muscle cells ( i.e., SMC HA and SMC Hl cells), endothelial cells (i.e., HUVEC and CPAE cells) fibroblast cells (i.e., HS 68 and MRC-5 cells), glioblastoma cells (i.e., U118 cells) and monocyte / macrophages (ie, THP-1 cells). Approximately 2 x 10 cells were inoculated one day before transduction by the adenovirus in plates of 24 multiple wells. Each well was then infected at an MOI of 1 with GV10 (i.e., comprising the natural adenovirus fiber protein) or with GV10 UTV (i.e. comprising the chimeric adenoviral fiber) in a volume of 250 μl for about one hour . The wells were then washed and incubated for two days, after which the lacZ activity of the cell lysates was determined. The results were reported as the average of duplicate measurements. As illustrated in Figure 9, the use of the GV10 UTV vector to transfer a reporter gene to a panel of cell lines confirms that the presence of the chimeric fiber protein (UTV) increases the distribution of the lacZ gene to cells expressing levels low or P1340 / 98MX undetectable fiber receptor (ie, receptor-minus cells) of about 5 up to about 300 fold compared to the wild type vector (GV10). In cells expressing high levels of the fiber receptor (i.e., receptor-plus cells), incorporation of the chimeric fiber protein into the GV10 UTV vector results in an increase in gene distribution up to about 3-fold. This reduction in expression observed with the transduction of the non-epithelial receptor-less cells compared to the epithelial cells of the receptor-plus by the adenovirus comprising a natural fiber protein (ie, GV10) correlates directly with the capacity relative of the vector to bind to these different types of cells, as reported in Example 3. These results report the view that the low expression of the receptor for the natural adenovirus fiber protein is a significant limiting factor to its efficient transduction by the current adenovirus vectors. Similarly, the ability of chimeric coat protein (ie, chimeric fiber protein) to enhance gene transfer in vivo was assessed. Three BALB / c mice were inoculated intranasally with approximately 1 x 108 pfu of GV10 P1340 / 98MX in 50 μl of a saline solution comprising 10 mM MgCl 2 and 10 mM Tris (pH 7.8). Three other mice received the same dose of GV10 UTV, and two mice received saline alone. The animals were sacrificed two days after the administration, and the lungs were assessed for lacZ activity. The lungs were prepared for analysis by rapidly freezing the lung in liquid nitrogen, laying the tissue with a mortar, and Using the ground tissue in 1.0 ml of the lacZ indicator lysis buffer (Promega Corp., Madison, Wl). A fluorometric assay was used to inspect the activity of lacZ, and the results of the experiments were reported as the average life activity for each group of animals. The results of these experiments are illustrated in Figure 10. As can be seen from this Figure, the in vivo gene transfer mediated by the vector GV10 UTV comprising the chimeric fiber protein (UTV) compared to the vector comprising the natural fiber protein (GV10) resulted in an average distribution 8 times larger than the mouse lung. These results confirm in this way that the incorporation of a chimeric cover protein (in this case, a chimeric fiber protein) into an adenoviral vector substantially increases the efficiency of the protein.

P1340 / 98MX vector-mediated gene distribution both in vivo and in vitro compared to an adenovirus vector comprising the natural fiber protein. In addition, the results support the conclusion that the low expression of the fiber receptor is a significant factor that contributes to the sub-optimal distribution observed in the lung and in other tissues. Also, the results confirm the superiority of the GV10 UTV vector, as well as other similar UTV vectors, over other adenoviral vectors, currently available for gene transfer (eg, distribution of the CFTR gene) to the lung and other tissues.

EXAMPLE 6 This example evaluates the ability of a vector according to the invention comprising a chimeric coat protein (e.g., a chimeric fiber protein) to interact with the average passenger DNA of a protein / DNA interaction, and to carry This mode the DNA in a cell in a way "carried to the shoulder". For these experiments, an adenoviral vector comprising the natural fiber (i.e., GV10) and an adenoviral vector comprising the chimeric fiber (i.e., GV10 UTV) were used to assess the gene transfer to the epithelial-receptor-plus cells (e.g. say, 293 cells, P1340 / 98MX A549, and H700 T). In the control experiments, the cells were transduced with the vectors as previously described. In the experimental condition, the vectors were incubated in the pGUS, which comprises a β-glucuronidase reporter gene, such that the chimeric adenorival fiber protein was able to become complex with the plasmid DNA. Specifically, approximately 5 x 10 active particles (i.e., units of fluorescence foci (ffu)) of GV10 or GV10 UTV were incubated for 1 hour for approximately 2.5 μg of the pGUS plasmid DNA. The mixed Q was then added to approximately 2 x 10 of the indicated cells in 250 μl of DMEM containing 10% fetal bovine serum. The activity of both β-glucuronidase and β-galactosidase was then assessed by fluorometric assay at 10 days after transduction. The expression of β-glucuronidase in the cells was inspected in a manner similar to the β-galactosidase assay for the expression of lacZ, by inspecting the generation of a blue color when the β-glucuronidase catalyses a reaction with the X-glu substrate. The results of these experiments are illustrated in Figure 11. Comparable levels of lacZ expression were obtained with either a GV10 vector (ie, comprising the natural fiber protein) or a GV10 UTV vector (i.e. it comprises the chimeric protein of P1340 / 98MX fiber) were used to transfer the reporter gene into cis epithelial cells. In comparison, the natural vector was able to intracellularly transfer the pGUS plasmid to only a relatively low level in all epithelial cells, as assessed by the expression of the β-glucuronidase gene. This basal level of gene transfer was probably achieved by means of receptor-mediated admission (RME) of the surrounding molecules, as previously described (PCT Patent Application WO 95/21259). However, with the use of a GV10 UTV vector comprising a chimeric fiber protein, the transfer of plasmid pGUS was substantially increased, in the case of gene transfer to 293 cells, the expression of β-glucuronidase directed by the plasmid pGUS exceeded the expression observed after the transfer mediated by the vector GV10 UTV of a reporter gene in the cis-linked. These results confirm that a vector comprising a chimeric coat protein such as a chimeric fiber protein according to the invention demonstrates an increased transfer of a nucleic acid which is not located in cis with the vector. Ostensibly, this improved gene transfer is effected by the occurrence of a protein / DNA interaction between the relatively charged residues in the fiber.

Chimeric P1340 / 98MX (eg, residues of the polylysine sequence), which result in binding to the nucleic acid vector; however, other means of improvement are also possible.

Example 7 This example describes the construction of additional plasmids containing UTV or UTV-like sequences in the C-terminus of the fiber protein. The transfer plasmid pl39 (F5 *) (Figure 12, also known as pl93NS (F5 *), pl93 (F5 *), and pAd NS 83-100 UTV) described in Example 2 and used as a starting point for the construction of these additional plasmids containing the chimeric proteins of adenovirus fiber. As depicted in Figure 12, the pl93 (F5 *) contains a mutated fiber gene with a BamHI site between the last codon of the fiber protein and the terminator codon of the fiber protein with change in the reading frame . Additional mutant transfer plasmids, constructed as described herein, contain sequences in the C-terminus of the fiber coding for a glycine / amino acid serine repeating linker, a targeting sequence, and a generalizing codon. These plasmids were made by cloning the synthetic oligonucleotides at the BamHI site of pl93 P1340 / 98MX (F5 *) to create the transfer plasmid pl93NS (F5 *) pGS (K7) (also known as pl93 (F5 *) pGS (K7) or pNS (F5 *) pK7) depicted in Figure 13. this way, the sequence of the natural Ad5 fiber gene is: TCA TAC ATT GCC CAA GAA TAA AAA AGAA [SEQ ID NO: 59] Ser Tyr He Ala Gln Glu [SEQ ID NO: 60] where "TAA" is a stop codon, and the polyadenylation sequence is animated. The C-terminus of the mutated fiber gene present in pl93 (F5 *) is: TCA TAC ATT GCC CAA GAA GGA TCC AATAAA GAA [SEQ ID NO: 19] Being Tyr He Wing Gln Glu Gly Being [SEQ ID NO: 20] wherein the underlined sequence indicates the mutated BamHI site introduced into the fiber protein, and the polyadenylation sequence is animated. In comparison, the amino acid sequence of the C-terminus of the fiber gene present in pl93NS (F5 *) pGS (K7) is: GSGSGSGSGS KKKKKKK [SEQ ID NO: 22] wherein the underlined sequence indicates the mutated BamHI site introduced into the fiber protein, and the animated sequence indicates the polylysine sequence at the C-terminus. This amino acid sequence is encoded by the nucleic acid sequence: GGA TCA GGA TCA GGT TCA GGG AGT GGC TCT AAA AAG AAG AAA AAG AAG AAG TAA [SEQ ID NO: 21], where P1340 / 98MX "TAA" is a stop codon. The overlapping synthetic oligonucleotides used to be the transfer plasmid p39S (F5 *) pGS (K7) were: pK7 (homosense), GA TCA GGA TCA GGT TCA GGG AGT GGC TCT AAA AAG AAG AAA AAG AAG AAA TAA G [SEQ ID NO : 61]; pK7a (antisense), GA TCC TTA CTT CTT CTT TTT CTT TTT AGA GCC ACT CCC TGA ACC TGA TCC T [SEQ ID NO: 62]. The homosense and antisense oligonucleotides were mixed in equimolar ratios and cloned into the BamHI site of pl93NS (F5 *) to create pl93NS (F5 *) pGS (Pk7). Verification of the insert correctly oriented in pl93NS (F5 *) pGS (pK7) was performed by PCR using the homosense primer pK7s and the antisense oligonucleotide primer in the 3 'direction A5a32938, CAGGTTGAATACTAGGGTTCT [SEQ ID NO: 63]. The plasmid was also verified to contain the insert correctly oriented by sequencing the DNA sequence in the region of the insert using primer A5a32938. The transfer plasmid pl93NS (F5 *) was used in the construction of additional mutant transfer plasmids which additionally contain a UTV cell targeting sequence or UTV-like sequence of the C-terminus of the fiber protein. These plasmids include pl93NS (F5 *) pGS (null) (also known as pl93 (F5 *) pGS (null) or pl93 (F5 *) pGS), pBSS P1340 / 98MX 75-100 pGS (nuil), pBSS 75-100 pGS (RK32), pBSS 75-100 pGS (RK33) and pBSS 75-100 pGS (tat). To construct p1393 (F5 *) pGS (null), the complementary overlapping oligonucleotides pGSs, GATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGTTAAA [SEQ ID NO: 64], and pGSa, GATCTTTAACTAGTCGAGCCACTGCCAGATCCTGAACCG [SEQ ID NO: 65] were constructed by direct ligation into the plasmid pl39NS (F5 * ) digested with BamHI. Verification of the correctly oriented clone was performed by PCR using the pGSs primer and the antisense oligonucleotide primer, in the 3 'direction, A5a32938. The plasmid was also checked to contain the insert correctly oriented by sequencing the DNA sequence in the region of the insert using primer A5a32938. The vector pBSS 75-100 pGS (null) (also known as pBSS 75-100ΔE3 pGS (nuil)) shown in Figure 14 was constructed by replacing the Nhel to Salí fragment from pBSS 75-100 with the corresponding fragment from p pl93NS (F5 *) pGS (null). The Spel site that is not within the chimeric fiber gene was then removed by partial restriction of the plasmid with Spel, filling in the Klenow fragment and then religating the vector. The resulting vector comprises the relevant nucleic acid sequence: GCCCAAGAAGGATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGTTAA [SEQ ID NO: 23], (wherein "TAA" is a codon of P1340 / 98MX termination, which codes for the amino acid sequence Ala Gln Glu Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Thr Ser [SEQ ID NO: 24]. Inserts of plasmids pBSS 75-100 pGS (RK32) (also known as pBSS 75-100? E3 pGS (RKKK) 2, or pBSS 75-100? E3 pGS (RKKK2)), pBSS 75-100 pGS (RK33) (also known as pBSS 75-100? E3 pGS (RKKK) 3 or pBSS 75-100? E3 pGS (RKKK3)), and pBSS 75-100 pGS (tat), were constructed by direct ligation in the pBSS 75-100 plasmid pGS (null) digested with Spel. To construct pBSS 75-100 pGS (RK32) depicted in Figure 15, the complementary overlapping oligonucleotides RK32s, CTAGAAAGAAGAAACGCAAAAAGAAGA [SEQ ID NO: 66] and RK32a, CTAGTCTTCTTTTTGCGTTTCTTCTTT [SEQ ID NO: 67], were employed. The resulting vector comprises the relevant nucleic acid sequence: GCCCAAGAAGGATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGAAAGAAGAAACGCAA AAAGAAGACTAGTTAA [SEQ ID NO: 25] (where "TAA" is a stop codon), which encodes the amino acid sequence Ala Gln Glu Gly Ser Gly Ser Gly Ser Gly Ser Thr Arg Lys Lys Lys Arg Lys Lys Lys Thr Ser [SEQ ID NO: 26]. To construct pBSS 75-100 pGS (RK33)) depicted in Figure 16, complementary overlapping oligonucleotides, RK33S, were employed, P1340 / 98MX CTAGAAAGAAGAAGCGCAAAAAAAAAAGAAAGAAGAAGA [SEQ ID NO: 68] and RK33a, CTAGTCTTCTTCTTTCTTTTTTTTTTGCGCTTCTTCTTCTTT [SEQ ID NO: 69]. The resulting vector comprises the relevant nucleic acid sequence: GCCCAAGAAGGATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGAAAGAAGAAGC GCAAAAAAAAAGAAAGAAGAAGACTAGTTAA [SEQ ID NO: 27] (where "TAA" is a stop codon), which encodes the amino acid sequence Ala Gln Glu Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Thr Arg Lys Lys Lys Arg Lys Lys Lys Arg Lys Lys Lys Thr Ser [SEQ ID NO: 28]. To construct pBSS 75-100 pGS (tat), the complementary superimposed oligonucleotides, TATs, CT AGT TAT GGG AGA AAA AAG CGC AGG CAA CGA AGA CGG GCA T [SEQ ID NO: 70] and TATa, CT AGA TGC CCG TCT were used TCG TTG CCT GCG CTT TTT TCT CCC ATA A [SEQ ID NO: 71]. The resulting vector comprises the relevant nucleic acid sequence: ACT AGT TAT GGG AGA AAA AAG CGC AGG CAA CGA AGA CGG GCA TCT AGT [SEQ ID NO: 72], which encodes the amino acid sequence Thr Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ser Ser [SEQ ID NO: 73]. Verification of the correctly oriented clone was performed by PCR using the primers (RK32s, RK33s, or TATs) for each of the three respective plasmids, and using the oligonucleotide primer, antisense, in the P1340 / 98MX 3 'address, A5a32938. Each of the plasmids were also checked to contain the insert correctly oriented by sequencing the DNA sequence in the region of the insert, using primer A5a32938.

EXAMPLE 8 This example describes the construction of plasmids containing the UTV domains in the loop handle. Plasmids that contain a UTV sequence (and / or a spacer sequence) in an exposed loop of the fiber protein are constructed by incorporating any of the sequences mentioned above (as well as any of the additional UTV-like sequences in the fiber protein. plasmid transfer vector pl93NS (F5 *) to construct the additional transfer vector pl93NS F5F2K (also called pl93 F5F2K) depicted in Figure 17. The plasmid pl93NS F5F2K contains a unique Spe I restriction site, with the gene of the Ad2 fiber coding for an exposed loop in the protein Specifically the protein gene present in pl93NS F5F2K comprises the fiber sequence: ATT ACÁ CTT AAT GGC ACT AGT GAA TCC ACÁ He Thr Leu Asn Gly Thr Ser Glu Ser Thr P1340 / 98MX GAA ACT [SEQ ID No: 29] Glu Thr [SEQ ID No: 30] wherein the underlined sequence indicates the new Spel site introduced into the fiber gene. This vector was then used to clone the targeting sequences in the Spel site. In particular, a nucleic acid sequence that codes for the stretch of 8 basic amino acids RKKKRKKK (Arg Lys Lys Lys Arg Lys Lys Lys [SEQ ID No.74]) comprising the heparin binding domain was cloned into the Spel site of pl93 F5F2K using the homosense and antisense oligonucleotides, superimposed. Specifically, (RKKK) 2, comprises, in part, the sequence: TCT AGA AAA AAA AAA CGC AAG AAG AAG ACT Thr Arg Lys Lys Lys Arg Lys Lys Lys Thr AGT [SEQ ID NO: 75] Ser [SEQ ID NO: 76].

The 27-mer homosentide oligonucleotide, RK32s and 27-mer antisense oligonucleotide RK32a, described in Example 7 were used to clone the sequence P1340 / 98MX PolyGS (RKKK) 2 comprising the peptide portion RKKKRKKK [SEQ ID NO: 74]. Plasmid pl93NS F5F2K (RKKK) 2 was constructed by cloning the DNA sequence encoding the binding domain at the Spel site of pl93NS F5FK2. The overlapping homosense and antisense oligonucleotides encoding the binding domain were first fixed and then ligated directly into the Spel restriction site to result in the plasmid pl93NS F6F2K (RKKK) 2 shown in Figure 18. This plasmid also it is known as pl93NS F5F2K (RKKK2), pl93NS F5F2K (RK32), or pl93 F5F2K (RKKK2). The relevant portion of the modified loop of the protruding (heavily enlarged chromosome stained chromosome) fiber present in pl93NS F5F2K (RKKK) 2 is: ATT ACÁ CTT AAT GGC ACT AGA AAG AAA CGC AAA AAG AAG He Thr Leu Asn Gly Thr Arg Lys Lys Lys Arg Lys Lys Lys ACT AGT GAA TCC ACÁ GAA ACT [SEQ ID NO: 31] Thr Ser Glu Ser Thr Glu Thr [SEQ ID NO: 32].

In addition, a sequence (RKKK) 3 or other variations of this sequence can be inserted into pl93NS F5F2K. This sequence comprises, in part: TCT AGA AAG AAG AAG CGC AAA AAA AAA AGA AAG AAG AAG Thr Arg Lys Lys Lys Arg Lys Lys Lys Arg Lys Lys Lys P1340 / 98MX ACT AGT [SEQ ID NO: 77] Thr Ser [SEQ ID NO: 78].

The sequence can be inserted with the use of the 39-mer homosense oligonucleotide (RKKK) 3 (s) (ie, comprising the CT sequence AGA AAG AAG AAG CGC AAA AAA AAA AGA AAG AAG AAG A [SEQ ID NO: 79 ]), and the 39-mer antisense oligonucleotide (RKKK) 3 (a) (ie, comprising the sequence CT AGT CTT CTT CTT TTT TTT TTT GCG CTT CTT CTT [SEQ ID NO: 80]). The resulting plasmid pl93NS F5F2K (RKKK) 3 is depicted in Figure 19. This plasmid is also known as pl93NS F5F2K (RKKK3), pl93 F5F2K (RKKK3), or pl93 F5FK [RK33]. The relevant portion of the modified loop of the protruding (heavily enlarged chromosome stained chromosome) fiber present in pl93 F2F2K (RKKK) 3 is: CTT AAT GGC ACT AGA AAG AAG AAG CGC AAA AAA AAA AGA AAG Leu Asn Gly Thr Arg Lys Lys Lys Arg Lys Lys Lys Arg Lys AAG ACT AGT GAA TCC ACÁ [SEQ ID NO: 33] Lys Thr Ser Glu Ser Thr [SEQ ID NO: 34] EXAMPLE 9 This example describes the construction of plasmids containing the base chimeric proteins of P1340 / 98 X penton comprising UTV or UTV-like sequences. The pACT (? RGD) transfer plasmid (also described as the pAT plasmid in US Patent No. 5,559,099) was derived, in part, by manipulating a plasmid containing the unique BamHI / Pmel fragment (13259-21561) of the Ad5 genome , and contains, among other things, a penton-based protein comprising a suppression of 8 amino acids that constitutes the av-integrin binding domain, and a substitution of the suppressed region for amino acids that constitutes a single Spel site, for the insertion convenient of exogenous sequences. To construct plasmid pACT (RKKK) 3, (also known as pACT (RKKK3) or pACT (RK33)) shown in Figure 20, the overlapping, complementary oligonucleotides, RK33s and RK33a were directly ligated into a pACT plasmid (? RGD) digested with Spel. Verification of the correctly oriented clone was performed by PCR using the primer RK33a for the plasmid and the oligonucleotide primer A5sl5002, homosentide, in the 5 'direction. The plasmid was also verified to contain the insert correctly oriented by sequencing the DNA sequence in the insert region using primer A5sl5002. The relevant portion of the UTV domain that will result in the penton-based chimeric protein in pACT (RKKK) 3 is: P1340 / 98MX AAC GAT ACT AGA AAG AAG AAG CGC AAA AAA AAA AGA AAG AAG AAG Asn Asp Thr Arg Lys Lys Lys Arg Lys Lys Lys Arg Lys Lys Lys ACT AGT GCC ACÁ [SEQ ID NO: 35] Thr Ser Ala Thr [SEQ ID NO: 36].

Plasmid pACT (RK32) (which can also be called pACT (RKKK2) or pACT (RKKK) 2) shown in Figure 21 can be constructed in a similar manner using the superimposed primers RK32s and RK32a. The relevant portion of the UTV domain present in the penton-based chimeric protein in pACT (RKKK) 2 is: AAC GAT ACT AGA AAG AAG AAG AGA AAG AAG AAG ACT Asn Asp Thr Arg Lys Lys Lys Arg Lys Lys Lys Thr AGT GCC ACÁ [SEQ ID NO: 37] Ser Ala Thr [SEQ ID NO: 38].

EXAMPLE 10 This example describes the construction of plasmids containing the sequence of UTV or UTV-like in the adenovirus exon protein, and in particular containing these sequences in an exposed loop of the adenovirus exon protein. These plasmids can be constructed by using P1340 / 98MX from another transfer plasmid, the pACT Hll plasmid, shown in Figure 22. The pACT Hll plasmid itself is derived from the pACT plasmid (comprising from 13259-21561 of the Ad5 genome), which contains the majority of the coding sequence of the exon protein, which corresponds to about 18842-21700). In particular, the pACT Hll can be constructed by incorporating an Xbal site in the region of loop 1 of the exon protein Ad5. In similar techniques to incorporate the Xbal site, or any other convenient restriction site, in the region of either loop 1 or loop 2, or in another exposed loop of the exon protein. Homosense and antisense primers can be used to amplify the loop region of the Ad5 DNA by PCR, and at the same time, introduce a mutation that results in a unique, mutated Xbal site in the loop region 1. In particular, the homosentide primer, GGACAGGGGCCCTACTTTTAAGCCCTACTCTGGCA [SEQ ID NO: 81], which contains the unique restriction site Apal, which occurs naturally, which is presented in pACT, and the antisense primer, ATCTTCACTGTACAATACCACTTTAGGAGTCAAGTTATCACCTCTAGATGCGGTCGCCT [SEQ ID NO: 82 ], which contains the unique restriction site, from BsrGI. The PCR product, which contains the Xbal site, can then be cut with BsrGI and Apal, which is cloned P1340 / 98MX again in pACT to replace the Apal fragment to BsrGI. The resulting plasmid, pACT Hll, contains a unique Xbal site for the insertion of the UTV sequence in loop 1 of the exon. The presence of the Xbal site in the clone pACT Hll can be verified by restriction digestion Xbal, which must linearize the plasmid. Part of the amino acid sequence of loop 1 of the non-mutated exon comprises the sequence TEATGNGDNL [SEQ ID NO: 83]. In comparison, the amino acid sequence of sequence 1 of the mutated exon, which follows the natural TEA residues, in pACT Hll (Figure 22) comprises the sequence TASRGDNL [SEQ ID NO: 40] (ie, encoded by the sequence of nucleic acid ACCGCATCTAGAGGTGATAACTTG [SEQ ID NO: 39]). The Xbal site of pACT Hll can then be used as a single site in which to clone the universal targeting sequences, such as RKKKRKKK (SEQ ID NO: 74), for example, using the overlapping oligonucleotides RK32s and RK32a. particular that results from these manipulations, i.e. pACT Hll (RKKK) 2 (or pACT Hll (RK32) or pACT Hll (RKKK2)) is depicted in Figure 23. This plasmid comprises the sequence: ACC GCA TCT AGA AAG AAA AAA CGC AAA AAG AAG ACT AGA Thr Ala Ser Arg Lys Lys Lys Arg Lys Lys Lys Thr Arg P1340 / 98MX GGT GAT AAC TTG [SEQ ID NO: 41] Gly Asp Asn Leu [SEQ ID NO: 42].

Other UTV or UTV-like sequences can also be cloned in the region of loop 1 in the exon protein, and / or in the loop region of the exon protein. A similar approach can be used to mutate the sequence encoding the exon loop 2 to make plasmid pACT H12 (not shown) that contains a unique restriction site (such as the Xbal site in which UTV sequences can be cloned). Also, plasmid pACT Hll (RKKK) 3 (or pACT Hll (RKKK3) or pACT Hll (RK33)) can be constructed using the complementary superimposed oligonucleotides RK33s and RK33a, and directly ligated to the PCR product in the plasmid pACT Hll, digested with Xbal Verification of the correctly oriented clone can be performed by PCR using the RK32 homosentide primer for the plasmid and an oligonucleotide primer, antisense, in the 3 'direction, appropriate The plasmid can be verified to contain the insert correctly oriented by sequencing the DNA sequence in the region of the insert using the 3 'antisense primer, similar approaches can be used for the construction of the Analog transfer vector, particularly the transfer vectors P1340 / 98MX analogs pACT H12 (RKKK), and pACT H12 (RKKK) 2.

EXAMPLE 11 This example describes the construction of the plasmid having a short tree fiber protein. In particular, this example describes the construction of the pl93 plasmid F5F9sK. The plasmid pl93 F5F9sK (also known as pl93 F5F9K-short) is presented in Figure 24. This vector encodes a chimeric fiber protein where about two thirds of the Ad5 fiber tree is deleted and the Ad5 fiber overhang is replaced with the Ad9 fiber outgoing. The plasmid pl93P5F9K-short was constructed from pl93NS (F5 *). The oligonucleotide primers GGACTAGTAG CATTTAATAA AAAAGAAGAT AAGCGC [SEQ ID NO: 84] and CCGGATCCTC ATTCTTGGGC GATATAGG [SEQ ID NO: 85] were used to amplify the Ad9 sequence encoding the last repeat and the branching of the fiber gene tree. The PCR product was then purified using normal techniques, digested with the restriction enzymes Nhel and BajnHI, which allowed the cloning of the PCR product in the Nhel / BamEl region of the transfer plasmid pl93NS (F5 *). The resulting short tree fiber protein can be used in the construction of adenoviral vectors P1340 / 98MX as described below, in addition, any one or more of the sequence of UTV or UTV-like mentioned above can be incorporated into the short-tree fiber, and the resulting fiber, can be employed for the distribution of cells.

EXAMPLE 12 This example describes the construction of plasmids containing UTV or UTV-like sequences in an extended structure, particularly in the exon and / or penton base protein, to result in exon and / or penton base proteins, elongated, which are therefore able to contact the cells and participate in the selection of cell targets. The resulting chimeric proteins were punctured, in the sense that they comprise an insertion of a non-native sequence of amino acids that will protrude from the surface of the virus. The primers lalfa (s), GGGCTGCAGGCGGCCGCAGAAGCTGAAGAGGCAGCCACACGGGCTGAGGAGAA [SEA ID NO: 86], and lalfa (a), GGGGTGCACACAGCTTCGGCCTTAGCGTTAGCCTGTTTCTTCTGAGGCTTCTCGACCT [SEQ ID NO: 87], can be used to amplify the region of the penton gene encoding the a-helical domain of 32 amino acids that follows in the sequence of RGD. This P1340 / 98MX sequence of 32 amino acids comprises the sequence of ATRAEEDRAEAEAAAEAAAPAAQPEVEKPQKK [SEO ID NO: 88]. The primers used can also code for an additional a-helical sequence at either end, such that, for example, the final amplified DNA sequence codes for the sequence: CTG CAG GCG GCC GCA GAA GCT GAA GAG GCC GCC ACÁ CGG GCT GAG Leu Gln Wing Wing Wing Glu Wing Glu Glu Wing Wing Thr Arg Wing Glu GAG AAG CGC GCT GAG GCC GAA GCA GCG GCC GAA GCT GCC GCC CCC Glu Lys Arg Wing Glu Wing Glu Wing Wing Wing Glu Wing Wing Pro GCT GCG CAA CCC GAC GTC GAG AAG CCT CAG AAG AAA CAG GCT AAC Ala Wing Gln Pro Glu Val Glu Lys Pro Gln Lys Lys Gln Wing Asn GCT AAG GCC GAA GCT GTG CAG GCG GCC GCA GAA GCT GAA GAG GCA Ala Lys Ala Glu Ala Val Gln Ala Ala Ala Glu Ala Glu Glu Ala GCC ACÁ CGG GCT GAG GAG AAG CGC GCT GAG GCC GAA GCA GCG GCC Ala Thr Arg Ala Glu Glu Lys Arg Ala Glu Ala Glu Ala Ala Ala GAA GCT GCC GCC CCC GCT GCG CAA CCC GAG GTC GAG AAG CCT CAG Glu Wing Wing Wing Pro Wing Wing Gln Pro Glu Val Glu Lys Pro Gln P1340 / 98MX AAG AAA CAG GCT AAC GCT AAG GCC GAA GCT GTG CAC [SEQ ID N0: 89] Lys Lys Gln Ala Asn Ala Lys Ala Glu Ala Val His [SEQ ID N0: 90] wherein the animated sequence corresponds to the non-penton sequence encoded by the primers, and to the underlined sequence represents the amino acids encoded by the two compatible, restriction sites, Sfcl and ApaLI. These amino acids also preserve the integrity of alpha analysis in accordance with normal computer programs designed to predict the structure of a -ysis. The PCR product encoding these amino acids can be cut with either Sfcl or ApaLI, religated, and then cut again with both enzymes. The site linkage similar to the similar site preserves the site for trimming; however, the ligation of compatible, but not similar, sites destroys the site. Therefore, in trimming the bound product, multiple fragments that are multiples of the original size of the PCR product will be produced. There will be completely trimmed fragments (approximately 150 bp), fragments of between about 300 bp (which have a destroyed restriction site), and the approximately 450 bp fragment (which P1340 / 98MX has 2 sites destroyed), and so on. The procedure therefore allows the original sequence coding for the 50 amino acids (lalfa) to be doublet (2alpha), triplet (3alpha), and so on, to clone a-helical, uninterrupted, large regions in the protein to create a greater "perforation" or extension of the protein. For example, the double 2alpha product (ie 2alpha2) can be cloned into the first PpulOl site of the plasmid pSPdelta (shown in Figure 25), to create the plasmid pSP2alpha (depicted in Figure 26). The plasmid pSPdelta is constructed from pUC19 base plasmid, or any other suitable cloning plasmid. The transfer plasmid pSPdelta can be cooled to make further modifications of the penton or exon protein which allows the UTV sequence (or any other targeting sequence) to rise from the surface of the virion. This elevation of the targeting surface in a perforated structure (eg, a "tower" type) will minimize the steric hindrance of the interaction of penton and exon with the cell surface or the fiber protein, and allow greater access of the penton and exon chimeric proteins to interact with the cell surface. The plasmid pSPdelta contains a site of P1340 / 98MX single Spel cloning for the incorporation of UTV sequences. The Spel cloning site is blunted on either side by the PpulOl cloning sites which allows the incorporation of the DNA sequences encoding the amino acids that will raise the UTV sequence from the virion surface. Plasmid pSPdelta was constructed by cloning into the unique Xbal site of overlapping oligonucleotides pUCl9 that are designed to be inserted directly from the Xbal site. In particular, the homosentide oligonucleotide is: CTAGAGCAGCTATGCATGAAGGGACTAGTGGAGAGATGCATGCAGCCT [SEQ ID NO: 91]. The antisense, complementary oligonucleotide is: CTAGAGGCTGCATGCATCTCTCCACTAGTCCCTTCATGCATAGCTGCT [SEQ ID NO: 92]. The oligonucleotides are mixed in equimolar ratios and cloned into the Xbal site of pUC19. The presence of the correct insert can be confirmed by sequencing through the insert region, and by cutting the PpulOl plasmid. The Xbal sites on either side of the insert will allow convenient removal of this section in these latter clones. Because there are two PpulOl sites in pSPdelta, the plasmid can be partially restricted in the PpulOl digestion so that only one individual site is cut. The ligation of the PCR product comprising the ApaLI and Sfcl sites in the PpulOl compatible site P1340 / 98MX will destroy the first site that PpulOl in plasmid and will also destroy ApaLI and Sfcl sites. These destroyed sites are represented in Figure 26 (showing the plasmid pSP2alpha2) by "j". A second doublet product can then be cloned into the remaining PpulOl site of the plasmid pSP2alpha to produce the plasmid pSP2alpha2 depicted in Figure 27 which contains a Spel site for insertion of the cloning of a UTV sequence, or other sequence of selection of objectives, similar. Computer analysis of the secondary structure of the anticipated 2alpha2 protein confirms that a complete a-helix will be present except for the region of the Spel cloning site. In this way, while pSPdelta comprises the sequence: TCT AGA GCA GCT ATG CAT GAA GGG ACT AGT GGA GAC ATG CAT GCA Ser Arg Ala Ala Met His Glu Gly Thr Ser Gly Glu Met His Ala GCC TCT AGA [SEQ ID NO: 43] Ala Ser Arg [SEQ ID NO: 44], pSP2alpha comprises the sequence: TCT AGA GCA GCT ATG CAG GCG GCC GCA GAA GCT GAA GAG GCA Ser Arg Ala Ala Met Gln Ala Ala Ala Glu Ala Glu Glu Ala P1340 / 98MX GCC AC CGG GCT GAG GAG AAG CGC GCT GAG GCC GAA GCA GCG Wing Thr Arg Wing Glu Glu Lys Arg Wing Glu Wing Glu Wing Ala GCC GAA GCT GCC GCC CCC GCT GCG CA CA CCC GAG GTC GAG AAG Ala Glu Ala Ala Ala Pro Ala Ala Gln Pro Glu Val Glu Lys CCT CAG AAG AAA CAG GCT AAC GCT AAG GCC GAA GCT GTG CAG Pro Gln Lys Lys Gln Ala Asn Ala Lys Ala Glu Ala Val Gln GCG GCC GCA GAA GCT GAA GAG GCC GCC ACÁ CGG CCT GAG GAG Ala Ala Ala Glu Ala Glu Glu Ala Ala Thr Arg Ala Glu Glu AAG CGC GCT GAG GCC GAA GCA GCG GCC GAA GCT GCC GCC CCC Lys Arg Wing Glu Wing Glu Wing Wing Wing Glu Wing Wing Pro GCT GCG CAA CCC GAG GTC GAG AAG CCT GAG AAG AAA CAG GCT Ala Ala Gln Pro Glu Val Glu Lys Pro Gln Lys Lys Gln Ala AAC GCT AAG GCC GAA GCT GTG CAT GAA GGG ACT AGT GGA GAG Asn Ala Lys Ala Glu Ala Val His Glu Gly Thr Ser Gly Glu ATG CAT GCA GCC TCT AGA [SEQ ID NO: 45] Met His Ala Ala Ala Ser Arg [SEQ ID NO: 46], and pSP2alpha2 comprises the sequence: P1340 / 98MX TCT AGA GCA GCT ATG CAG GCG GCC GCA GAA GCT GAA GAG GCA GCC Be Arg Ala Ala Met Gln Ala Ala Ala Glu Ala Glu Glu Ala Ala ACÁ CGG GCT GAG GAG AAG CGC GCT GAG GCC GAA GCA GCG GCC GAA Thr Arg Ala Glu Glu Lys Arg Ala Glu Ala Glu Ala Ala Ala Glu GCT GCC GCC CCC GCT GCC CAA CCC GAG GTC GAG AAG CCT CAG AAG Ala Ala Ala Pro Ala Ala Gln Pro Glu Val Glu Lys Pro Gln Lys AAA CAG GCT AAC GCT AAG GCC GAA GCT GTG CAG GCG GCC GCA GAA Lys Gln Ala Asn Ala Lys Ala Glu Ala Val Gln Ala Ala Ala Glu GCT GAA GAG GCC GCC ACA CGG GCT GAG GAG AAG CGC GCT GAG GCC Wing Glu Glu Wing Wing Thr Arg Wing Glu Glu Lys Arg Wing Glu Wing GAA GCA GCG GCC GAA GCT GCC GCC CCC GCT GCG CAA CCC GAG GTC Glu Wing Wing Wing Glu Wing Wing Pro Wing Wing Gln Pro Glu Val GAG AAG CCT CAG AAG AAA CAG GCT AAC GCT AAG GCC GAA GCT GTG Glu Lys Pro Gln Lys Lys Gln Wing Asn Wing Lys Wing Glu Wing Val CAT GAA GGG ACT AGT GGA GAG ATG CAG GCG GCC GCA GAA GCT GAA His Glu Gly Thr Ser Gly Glu Met Gln Ala Ala Ala Glu Ala Glu P1340 / 98MX GAG GCC GCC ACA CGG GCT GAG GAG AAG CGC GCT GAG GCC GAA GCA Glu Wing Wing Thr Arg Wing Glu Glu Lys Arg Wing Glu Wing Glu Wing GCG GCC GAA GCT GCC GCC CCC GCT GCG CA CA CCC GAG GTC GAG AAG Ala Ala Glu Ala Ala Ala Pro Ala Ala Gln Pro Glu Val Glu Lys CCT CAG AAG AAA CAG GCT AAC GCT AAG GCC GAA GCT GTG CAG GCG Pro Gln Lys Lys Gln Ala Asn Ala Lys Ala Glu Ala Val Gln Ala GCC GCA GAA GCT GAA GAG GCA GCC ACÁ CGG GCT GAG GAG AAG CGC Wing Wing Glu Wing Glu Glu Wing Wing Thr Arg Wing Glu Glu Lys Arg GCT GCC GCC GAA GCA GCG GCC GAA GCT GCC GCC CCC GCT GCS CAA Wing Glu Wing Glu Wing Wing Wing Glu Wing Wing Pro Wing Wing Gln CCC GAG GTC GAG AAG CCT CAG AAG AAA CAG GCT AAC GCT AAG GCC Pro Glu Val Glu Lys Pro Gln Lys Lys Gln Ala Asn Ala Lys Ala GAA GCT GTG CAT GCA GCC TCT AGA [SEQ ID NO: 47] Glu Ala Val His Ala Ala Ser Arg [SEQ ID NO: 48].

UTV-like or UTV-like selection sequences can be cloned into the Spel site of plasmid pSP2alpha2. In particular, the overlapping oligonucleotides RK32s and RK32a can be cloned on site P1340 / 98MX Spel to create pSP2alfa2 (RKKK) 2, (or, pSP2alfa2 (RK32) or pSPSalfa2 (RKKK2)). The plasmid pSP2alpha2 (RKKK) 3, (o, pSP2alpha2 (RK33) or pSPSalpha2 (RKKK3)) can be constructed in a similar manner. Alternatively, the a-helical domain of whole 2alpha2 can be removed from the plasmid by restriction with Xbal and cloned into the Spel compatible site of pACT (? RGD) to create pACT 2alpha2 (RKKK) 2, (which can also be called pACT 2alfa2 (RKKK2) or pACT 2alpha2 (RK32)). Similar techniques can be used to produce pACT 2alpha2 (RKKK) 3, (which can also be called pACT 2alpha2 (RKKK3) or pACT 2alpha2 (RK33)). Similarly, chimeric exon proteins that are drilled (ie, comprise sequences that result in their extension) can be constructed by cloning the a-helical 2alpha2 domain at the Xbal site of pACT Hll to create pACT Hll 2alpha2 (RKKK ) 2 (which can also be called pACT Hll 2alfa2 (RKKK2) or pACT Hll 2alfa2 (RK32)). Similar techniques can be used to produce pACT H12 2alpha2 (RKKK) 2 (which can also be called pACT H12 2alpha2 (RKKK2) or pACT H12 2alpha2 (RK32)).

EXAMPLE 13 This example describes the construction of additional adenoviral vectors, in addition to those previously described that contain UTV or UTV-like sequences.

P1340 / 98MX in the adenoviral fiber protein. The construction of adenovirus vectors containing UTV modifications in the fiber can be accomplished in multiple ways by those skilled in the art. One method for creating the fiber vectors from plasmids described above is to first linearize the plasmid DNA with Sa2I and then transfect this DNA into a 293 cell line that was infected just prior to transfection with an E deleted adenovirus. . Adenovirus vectors deleted from E4 are unable to replicate in cell lines such as cell line 293, which only provides the El regions of the adenovirus in trans. The recombination in plasmid containing the modified fiber gene and the E4 regions with the deleted DNA in E4 results in an E4-containing vector, competent in the replication having the modified fiber gene. Accordingly, the plasmids pl93NS (P5 *) pGS (K7), pBSS 75-100 pGS (nuil), pBSS 75-100 pGS (RKKK) 2, pBSS 75-100 pGS (RKKK) 3, pBSS 75-100 pGS ( tat), pl93NS F5F2K (RK32), and pl93 P5F9sK were each linearized with SalI and transfected into infected 293 cells 1 hour before either the deleted E4 adenovirus vector, GVllA.Z (having the LacZ gene under control). of a cytomegavirus (CMV) promoter, or GVllA.S (which has a P1340 / 98MX alkaline phosphatase secretory gene under the control of a CMV promoter) the resulting adenovirus vectors, AdZ.F (pK7), AdZ.F (pGS), AdZ. F (RKKK) 2, (also known as AdZ.F (RKKK2) or AdZ.F (RK32)), AdZ. F (RKKK) 3, (also known as AdZ.F (RKKK3) or AdZ.F (RK33)), AdZ. F (tat), AdZ.F5F2K (RKKK) 2 (also known as AdZ, F5F2K (RKKK2) or AdZ.F5F2K (RK32)), and AdZ.F5F9sK (also known as AdZ, F5F9K-short) were obtained and purified through two successive rounds of plating of 293 cells. All vectors were verified to contain the correct sequence through PCR through the region of the insert, and by restriction analysis of the viral DNA obtained from the cells 293 infected with the vector by Hirt extraction. Western analysis (as previously described) was also used to examine the size of the protein, if desired. Western analysis of the fiber protein of the vector particles and / or lysates of infected electrophoresis cells in a polyacrylamide gel should show a corresponding change in the mobility of the fiber protein compared to the unmodified fiber protein that is consistent with the presence of additional amino acid sequences. For example, the Western analysis of the AdZ.F particles (pK7) verified that their fiber protein was changed upwards compared to that of the AdZ vector thatP1340 / 98MX comprises unmodified fiber, consistent with the presence of additional amino acids in the fiber protein AdZ.F (pK7). Other plasmid maps represented herein can be made, similarly, in adenoviral vectors by using the same procedure outlined above (or minor variations thereof).

EXAMPLE 14 This example describes the construction of adenoviral vectors containing UTV or UTV-like sequences in the adenoviral base protein of the penton. The method for making an adenoviral vector comprising a penton base protein is described for example in Wickham et al., J. Virol., 70, 6831-6838 (1996). A pACT vector described above containing the chimeric penton base protein (e.g., the transfer plasmids pACT 2alpha2 (RKKK) 2, pACT H12 2alpha2 (RKKK) 2, and pACT (? RGD)) can be digested, for example, with Bamñl, to linearize the plasmid. The Ad5 DNA can be digested with the restriction endonuclease Xmnl, which cuts the natural Ad5 at positions 14561 and 15710 within the Ad5 genome. The two largest fragments are purified from the smallest piece of 1 kb, then P1340 / 98MX are transfected together with the linearized plasmid into the appropriate cell line (e.g., a 293 cell line) to produce the recombinant virus. The adenoviral vectors produced in this way are purified from the unmodified vectors of potential contamination through two successive rounds of plaque purification in 293 cells. The resulting vectors are then verified to contain the correct sequence in the penton base region through restriction analysis of viral DNA obtained after Hirt extraction of 293 cells infected with the vector. The sequencing of the PCR products generated by amplifying the insert region from the viral DNA can also be used to verify the presence of the insert. Western analysis of the chimeric penton base subjected to electrophoresis in a polyacrylamide gel should show a corresponding change in the mobility of the penton base in comparison to the unmodified penton base which is consistent with the presence of the additional sequences of amino acids in the chimeric protein.

EXAMPLE 15 This example describes the construction of adenoviral vectors containing UTV or similar sequences to P1340 / 98MX UTV in the exon protein. To build the AdZ virus. H (RKKK) 2, the left and right arms of the vector are prepared to contain overlapping DNA sequences and recombine on either side of the pACT H11 (RK32) sequence from Pmel to BamHI to create an AdZ genome. H (RK32) intact. To construct the left arm, the purified Ad5 DNA is digested by restriction with Agel, which cuts Ad5 at positions 14499, 15283, 19017, 23063, 23656, 23411, and 31102. The fragment 0-14499 can then be used as the left arm and is purified from the other fragments by gel electrophoresis. The right arm can be prepared by digesting Ad5 DNA with DrdI. DrdI against Ad5 at positions 5458, 7039, 14004, 15593, 17257, and 21023. The 21023-35938 bp fragment can then be used as the right arm and purified from the other fragments by gel electrophoresis. These two fragments are then transfected with the Pmel / BamRI fragment of pACT H11 (RK32) in 293 cells. The Pmel cuts at position 13258 in Ad5, and BamHI cuts position 21562 in Ad5. Similar techniques can be used to produce AdZ.H (RKKK) 3.

EXAMPLE 16 This example describes the construction of vectors P1340 / 98MX that contain a short tree fiber protein. The method described herein for the construction of an adenovirus from the pl93 transfer plasmid F5F9Kcorto can be used in a similar manner for the construction of other adenoviral vectors from other short-tree fibers. Specifically, the p93 transfection plasmid F5F9Kcorto, which contains the essential E4 region of the adenovirus, was cut with Sali and transfected into 293 cells, which were infected one hour earlier with the AdSE.E4Gus adenoviral vector. The AdSE.E4Gus lacks the E4 region of the adenoviral genome and can replicate in 293 cells in the absence of complementation for the E4 genes. In this way, only when the AdSE.E4Gus DNA is recombined with the plasmid DNA pl93 F5F9K-short to obtain the E4 genes is the vector capable of replicating in 293 cells. During this recombination event, the newly formed vector also acquires the mutated fiber protein encoding the sequences encoded by the plasmids. The viable recombinant E4 + adenoviruses containing the F5F9Kcorto fiber chimera were then isolated by plating the transfected cell lysates, 5 days after transfection. The resulting vector AdSE. F5F9Kcorto was isolated and purified by normal virulogics comprising two successive rounds of plating in 293 cells.

P1340 / 98MX vector was verified to contain the correct insert by PCR and viral DNA restriction analysis. Oligonucleotide primers, which are primed on either side of the fiber gene, confirmed that the PCR product was the correct size for that encoded by a chimeric, cut fiber gene. Restriction analysis of the vector DNA showed that the new vector contained the correct restriction sites that are unique to the Ad9 fiber overhang.

EXAMPLE 17 This example describes the constructions of adenoviral vectors containing short tree fiber proteins and penton-based chimeric proteins that incorporate UTV or UTV-like sequences. For this construction, the viral DNA of AdS.F9sK can be suggested with Xmnl, as described above. Plasmid pACT H11 (RK32) is then cut with the restriction enzymes, Pmel and BamHI. The restriction-digested viral and plasmid DNAs are purified and transfected into 293 cells. The resulting vectors are isolated by successive rounds of plaque purification in 293 cells, and verified to contain the correct sequence in the base region of the penton. Restriction analysis of viral DNA obtained from 293 cells infected with the vector P1340 / 98MX by Hirt extraction. The sequencing of the PCR products generated by amplifying the insert region from the viral DNA can also be used to verify the presence of the insert. Western analysis of the penton-based chimeric protein in a polyacrylamide gel should show a corresponding change in the mobility of the chimeric base of the penton compared to the unmodified base of the penton which is consistent with the presence of non-native sequences of amino acids, additional (and the absence of native amino acid sequences). Other plasmids for which it correlates and which are not capable of being made in an adenoviral vector are presented herein, they can be made by using the same version or a slightly modified version of the procedure summarized above. In particular, the short tree fiber protein can be incorporated into an adenovirus having a "perforated" penton chimeric base protein that can optionally also incorporate a UTV or similar UTV sequence.

EXAMPLE 18 This example describes the construction of adenoviral vectors containing short tree fiber proteins and chimeric exon proteins that incorporate the P1340 / 98 X UTV sequences or similar to UTV. Viral DNA can be isolated from a short-tree vector such as AdZ.F9sK and cut with the restriction enzymes described above to make vectors comprising the exon chimeric proteins containing UTV. All other steps are the same as described, for example, in Example 17. The resulting vector must contain the short-tree fiber protein and the chimeric exon protein that incorporates the UTV or UTV-like sequences. In addition, this method can be cooled, a variety of transfer plasmids comprising different chimeric exon proteins. In particular, the short tree fiber protein can be incorporated into an adenovirus having a chimeric "perforated" exon protein that can optionally also incorporate a UTV or similar UTV sequence.

EXAMPLE 19 This example describes an evaluation of vectors, particularly adenoviral vectors, according to the invention, which comprise UTV or UTV-like sequences. For example, to confirm that the addition of the UTV or UTV-like sequences has no effect on P1340 / 98MX virus assembly, the kinetics of virus growth of vectors can be assessed. As representative of a plasmid containing UTV sequences, the growth behavior of pAd.F (pK7) was inspected and compared to that of the natural adenovirus (Ad5), as well as the AdZ adenoviral vector. F (RGD), which contains an insertion of a portion of the RGD peptide present in the sequence SACDCRGDCFCGTS [SEQ ID NO: 93]. For these studies, 293 cells were infected at a multiplicity of infection of 5 active virus particles / cell in either Ad5, AdZ. F (RGD) or AdZ.F (pR7), and the number of infectious particles (Fluorescent Focus Units (FFU)) produced by cells was determined after collection of the cells at 1, 2, and 3 days after the infection. The titles of AdZ. F (RGD) or AdZ.F (pK7) were somewhat lower, but not grammatically different from the Ad5 titer. As you can see in Figure 28, the maximum AdZ titles. F (RGD) and AdZ.F (pK7) were 80% and 56%, respectively, than Ad5. These results confirm that the growth kinetics of the two vectors was not substantially affected by the addition of the sequences, particularly a UTV or similar sequence of UTV, at the end of the fiber protein. The results also suggest that additional vectors comprising UTV or UTV-like sequences will not exhibit behavior P1340 / 98MX abnormal growth. In addition, vectors containing UTV or UTV-like sequences can be evaluated for their ability to bind to cells or distribute genes that are to be inhibited by negatively charged molecules (eg, heparin, heparan sulfate, chondroitin sulfate, etc.). .), covering, adenoviral, soluble proteins, or by pretreating cells with agents, (eg, chondroitinase, heparinase, sialidase, etc.) which cleave these negatively charged portions (see, for example, Wickham et al., Nature Biotechnology, 14, 1570-1573 (1996), as well as the preceding Examples). Soluble fiber protein will not inhibit most binding of a UTV vector to a cell (Wickham et al (1996), supra). These results suggest that the incorporation of UTV or UTV-like sequences in the penton, exon or fiber will not impart the ability of a recombinant adenovirus containing the coated chimeric protein to effect gene delivery. Also, in vivo infection studies, or in vitro transfections made in the presence of whole blood, can be used to confirm that the UTV vectors of the present invention are not limited for systemic distribution due to the saturation of the polycations. in recombinant adenoviruses with P1340 / 98MX polyanions in the blood. In the case that this binding prevents the capacity of the virus in particular for the transduction of target cells, the virus can be administered in a higher dose, preferably with the prohibition that is made to reduce any immune response associated with this type of high dose ( for example, administration of another serotype of the adenoviral vector, or techniques described in PCT International Application WO 96/12406; Mastrangeli et al., Human Gene Therapy, 7, 79-87 (1996)). All references cited herein, including patents, patent applications and publications, are thus incorporated in their entirety with reference to the same degree as if each reference were set forth in its entirety herein. While this invention has been described with emphasis on preferred embodiments, it will be appreciated by those skilled in the art that preferred embodiments may be prepared and used and that the invention may be practiced otherwise than as specifically described in the present. The present invention is proposed to include these variations and alternative practices. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims. P1340 / 98MX LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: GenVec. Inc. (ii) TITLE OF THE INVENTION: VECTORS AND METHODS FOR GENE TRANSFER TO CELLS (iii) NUMBER OF SEQUENCES: 12 (iv): CORRESPONDENCE ADDRESS: (A) RECIPIENT: Leydig, Voit & Mayer, Ltd. (B) STREET: Two Prudential Plaza, Suite 4900 (C) CITY: Chicago (D) STATE: IL (E) COUNTRY: USA (F) POSTAL CODE: 60601 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.20 P1340 / 98MX (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT (B) SUBMISSION DATE: Nov 27, 1996 (vii) DATA FROM THE PREVIOUS APPLICATION (A) APPLICATION NUMBER: US 08 / 563,368 (B) SUBMISSION DATE: 28-Jun-1995 (C) CLASSIFICATION: (viii) INFORMATION OF THE AGENT / LAWYER (C) ORDER NUMBER / REFERENCE: 74862 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION FOR SEQ. ID No: 1: P1340 / 98MX Lys Lys Lys Lys Lys Lys Lys Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION FOR SEQ. ID No: 2 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear P1340 / 98MX (ii) TYPE OF MOLECULE: peptide (ix) FEATURE: (C) OTHER INFORMATION: Xaa is Lys or Arg (xi) DESCRIPTION FOR SEQ. ID No: 3: Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION FOR SEQ. ID No: 4: Gly Ser Asn Lys Glu Ser Phe Val Leu Lys Lys Lys Lys Lys Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 5 P1340 / 98MX (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION FOR SEQ. ID No: 5: Wing Gly Being Asn Lys Asn Lys Glu Being Phe Val Leu Lys Lys Lys 1 5 10 15 Lys Lys Lys (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) P1340 / 98MX (xi) DESCRIPTION FOR SEQ. ID No: 6: GGA TCC AA 8 Gly Ser 1 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR SEQ. ID No: 7: GGA TCC AAT AAA GAA TCG TTT GTG TTA TGT 30 Gly Ser Asn Lys Glu Ser Phe Val Leu Cys 1 5 10 (2) INFORMATION FOR SEQ ID NO: P1340 / 98MX (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR SEQ. ID No: 8: GCC GGA TCC AAC AAG AAT AAA GAA TCG TTT GTG TTA 36 Wing Gly Being Asn Lys Asn Lys Glu Being Phe Val Leu 1 5 10 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION FOR SEQ. ID No: 9: P1340 / 98MX TATGGAGGAT CCAATAAAGA ATCGTTTGTG TTATGTTTGA ACGTGTTTAT TTTTC 55 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION FOR SEQ. ID No: 10: AATTGAAAAA TAAACACGTT GAAACATAAC ACAAACGATT CTTTATTGGA TCCTCCA 57 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear P1340 / 98MX (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION FOR SEQ. ID No: 11: TCCCCCCGGG TCTAGATTAG GATCCTTCTT GGGCAATGTA TGA 43 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (synthetic) (xi) DESCRIPTION FOR SEQ. ID No: 12: CGTGTATCCA TATGACACAG A 21 P1340 / 98MX

Claims (25)

1. CLAIMS 1. A chimeric adenovirus coat protein comprising a non-native sequence of amino acids, inserted into or instead of an internal coat protein sequence, wherein the chimeric adenovirus coat protein effectively binds to a range A broader range of eukaryotic cells than a natural adenovirus coat protein and wherein the chimeric adenovirus coat protein is not selective for a specific type of eukaryotic cell.
2. 2. A chimeric adenovirus coat protein comprising a non-native sequence of amino acids inserted into or instead of an internal coat protein sequence, wherein the chimeric adenovirus coat protein is effectively bound to an epithelial cell, a smooth muscle cell, an endothelial cell, a fibroblast cell, a glioblastoma cell and a monocyte cell.
3. 3. A chimeric adenovirus coat protein comprising a non-native amino acid sequence inserted into or instead of an internal coat protein sequence, wherein the chimeric adenovirus binds effectively to most all eukaryotic cells. P1340 / 98MX
4. 4. A chimeric adenovirus coat protein comprising a non-native amino acid sequence, wherein the chimeric adenovirus coat protein binds to SMC HA cells, HuVec cells, CPAE cells, HS 68 cells, MEC-5 cells, U118 cells and THP-1 cells more efficiently than a natural serotype fiber protein.
5. 5. The chimeric adenovirus coat protein according to any of claims 1-4, wherein the chimeric adenovirus coat protein effectively binds to substantially all eukaryotic cells.
6. 6. A chimeric adenovirus coat protein comprising a non-native amino acid sequence, wherein the chimeric adenovirus coat protein effectively binds to a present portion of the surface of the majority of all eukaryotic cells.
7. 7. The chimeric adenovirus coat protein according to claim 5, wherein the portion is a negatively charged portion. The chimeric adenovirus coat protein according to claim 6, wherein the chimeric adenovirus coat protein binds to at least a portion of the cell surface, selected from P1340 / 98MX from a group consisting of heparin, heparin sulfate, hyaluronic acid, dermatan sulfate, sialic acid, keratin sulfate and chondroitin sulfate. 9. The chimeric adenovirus coat protein according to claim 6, wherein the portion is heparin sulfate. 10. The chimeric adenovirus coat protein according to claim 6 or 7, wherein the portion is present in substantially all eukaryotic cells. The chimeric adenovirus coat protein according to any of claims 1 to 10, wherein the non-native amino acid sequence comprises three or more positively charged amino acid residues. The chimeric adenovirus coat protein according to any of claims 1 to 11, wherein the non-native amino acid sequence is an exposed loop of the protein. 13. A chimeric adenovirus coat protein according to any of claims 1 to 12, comprising a native amino acid sequence and a non-native amino acid sequence with carboxy terminal groups having from 3 to about 30 polylysine amino acid residues. 14. Chimeric coat protein from P1340 / 98MX adenovirus according to claim 13, wherein the native amino acid sequence comprises a deletion of at least one amino acid. 15. The chimeric adenovirus coat protein according to any of claims 1 to 14, wherein the non-native amino acid sequence comprises a spacer sequence. 16. The chimeric adenovirus coat protein according to any of claims 1 to 15, wherein the non-native sequence comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID N0: 5, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 90, and SEQ ID NO: 93. 17. The chimeric adenovirus coat protein according to claim 16, wherein the non-native amino acid sequence comprises SEQ ID NO: 4 or SEQ ID NO: 5 and wherein the non-native amino acid sequences comprise from about 3 to P1340 / 98MX approximately 30 lysine residues. 1
8. 8. The chimeric adenovirus coat protein according to any of claims 1 to 17, wherein the adenovirus chimeric protein is a fiber protein. 1
9. 9. The chimeric adenovirus coat protein according to any of claims 1 to 18, wherein the chimeric adenovirus coat protein is an exon or penton protein. 20. A vector comprising or coding for the coat protein according to any of claims 1 to 19. The vector according to claim 20, wherein the vector is a viral vector selected from the group consisting of non-virus. wrapped. 22. The vector according to claim 21, wherein the vector is an adenoviral vector. 23. The vector according to any of claims 20 to 22, wherein the vector comprises a passenger gene. 24. A host cell comprising a vector according to any of claims 20 to 23. 25. A method for genetically modifying a cell comprising contacting the cell with a vector according to any of claims 19 to 22. P1340 / 98MX