WO1994017832A1 - Ciblage et transport de genes et d'agents antiviraux dans des cellules par l'adenovirus penton - Google Patents

Ciblage et transport de genes et d'agents antiviraux dans des cellules par l'adenovirus penton Download PDF

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WO1994017832A1
WO1994017832A1 PCT/US1994/001263 US9401263W WO9417832A1 WO 1994017832 A1 WO1994017832 A1 WO 1994017832A1 US 9401263 W US9401263 W US 9401263W WO 9417832 A1 WO9417832 A1 WO 9417832A1
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cells
protein
penton
adenovirus
dna
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Glen R. Nemerow
Thomas J. Wickham
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Scripps Research Institute
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to gene therapy. * In particular, therapeutic agents and methods useful in targeting and delivering genes to particular cells ' , are disclosed, wherein adenovirus penton (a complex of
  • penton base and fiber or penton base is used to facilitate targeting and delivery.
  • a molecular conjugate consisting of a ligand for a cell surface receptor (i.e. transferrin) is covalently linked to a DNA binding moiety (i.e., poly L-lysine) (Wu, et al. , J__. 30 Biol. Chem. 262: 4429-4432 (1987); Wagner, et al. , PNAS USA 87: 3410-3414 (1990); and Wagner, et al. , PNAS USA 88: 4255-4259 (1991)).
  • a DNA binding moiety i.e., poly L-lysine
  • the conjugate is thus , able to bind to the cell receptor and is subsequently internalized by endocytosis. In many cases, however, 4 35 this approach is inefficient due to the targeting of the DNA conjugate to lysosomes (Zenke, et al. , PNAS USA 87: 3655-3659 (1990) ; Cotten, et al. , PNAS USA 87: 4033-4037 (1990) ) . Although lysosomatropic agents can sometimes be successfully used to inhibit this process, a high degree of variability is often observed in different cell types. (See, e.g., Curiel, et al., PNAS USA 88: 8850-8854 (1991) .)
  • adenovirus a human DNA virus which readily infects epithelial cells (Horwitz, "Adenoviridae and their replication", in Virology, Fields and Knipe, eds., Raven Press, NY (1990) pp. 1679-1740) . Since adenovirus efficiently disrupts the membranes of endocytic vesicles, co-internalization of the virus with the DNA conjugate allows rapid transfer of the conjugate into the cell cytoplasm before it can be subjected to lysosomal degradation.
  • adenovirus exhibits selective tropism for epithelial cells has also been exploited to reconstitute these cells in vivo with the human cystic fibrosis transmembrane conductance regulator (CFTR) (Rosenfeld, et al., Cell 68: 143-155 (1992)) and the alpha 1-antitrypsin genes (Rosenfeld, et al . , Science 252: 431-434 (1991) ) .
  • CTR cystic fibrosis transmembrane conductance regulator
  • Adenovirus type 2 has also been observed to facilitate the transfer of growth factors or toxins from endosomes into the cytoplasm of epithelial cells. (See, e.g., Fitzgerald, et al .
  • adenovirus-mediated gene therapy includes the efficient targeting of genes to epithelial cells, and the efficient delivery of genes to the cytoplasm, thus reducing lysosomal degradation of DNA. Further, the use of replication-defective strains of adenovirus reduces the cytopathic effect of replicating virus. Use of adenovirus may also facilitate the transfer of DNA into host cells. In vi tro, adenovirus augments the delivery of transferrin-poly L-lysine/DNA conjugates into host cells. (See, e.g. Curiel, et al., PNAS USA 88:
  • alpha 1-antitrypsin and cystic fibrosis transmembrane conductance regulator protein genes have been delivered into epithelial cells using adenovirus type 5 (Rosenfeld, et al. , Science 252: 431-434 (1991); Rosenfeld, et al. , Cell 68 . : 143-155 (1992) ) .
  • adenovirus penton or penton base independent of the rest of the adenovirus genome, facilitates the transfer of exogenous or non-native genes into recipient cells.
  • adenovirus penton or penton base for gene therapy over intact adenovirus or replication-deficient adenovirus, particularly in humans.
  • the adenovirus penton complex likely possesses all of the functional properties required for gene therapy including binding to epithelial cell receptors, stimulation of endocytosis, and penetration of endocytic vesicles.
  • large amounts of recombinant penton base and fiber may be produced in insect cells via the use of an expression system such as the baculovirus expression system. The proteins so produced are capable of assembling into the penton complex.
  • Chimeric forms of recombinant penton complex may also be constructed with the capacity to target foreign genes to non-epithelial cells. These chimeric forms are also capable of evading recognition and attack by the host's immune system. Furthermore, the use of penton complex and fiber is a much safer alternative for gene therapy applications than the introduction of adenovirus genes into mammalian cells as some forms of adenovirus contain genes that can transform cells. Removal of those deleterious genes from an adenovirus vector may not in itself be enough to prevent transformation as the vector may combine with a natural virus to produce a more pathogenic strain. The use of recombinant or conventionally purified penton base or penton complex of adenovirus overcomes these secondary in vivo events.
  • the present invention contemplates a therapeutic agent capable of specifically targeting epithelial cells comprising an adenovirus penton base operatively linked to a nucleotide sequence encoding a mammalian polypeptide and an active promoter.
  • the nucleotide sequence is conjugated directly to a region of the penton which does not alter its functional properties.
  • the nucleotide sequence may be "sense” or "antisense” .
  • the penton base or penton complex is incorporated into liposomes which contain the foreign therapeutic nucleotide sequence or antisense oligonucleotides.
  • the invention also contemplates the penton-mediated delivery of plasmid-based vectors containing the gene of interest under the control of strong constitutive or inducible promoters and/or enhancer elements to obtain higher levels of gene expression than that obtained by incorporating the target gene into the intact adenovirus genome.
  • the present invention contemplates a composition designed to specifically target epithelial cells and deliver a therapeutic nucleotide sequence to the cells, comprising an adenovirus-derived protein and the nucleotide sequence.
  • the protein is selected from the group consisting of penton base and penton complex.
  • the protein may be conventionally-purified or recombinant, and may further be derived from adenovirus type 2.
  • the present invention further contemplates that a composition may further comprise a pharmaceutically acceptable carrier or excipient.
  • the adenovirus- derived protein may be a complete protein, an apoprotein, a polypeptide, or an amino acid residue sequence comprising three or more amino acid residues linked together via peptide linkages.
  • the adenovirus-derived protein is biologically active, and is preferably non-toxic.
  • the therapeutic nucleotide sequence is operatively linked to the adenovirus-derived protein.
  • the therapeutic nucleotide sequence and the adenovirus-derived protein are contained within a liposome.
  • the nucleotide sequence encodes a polypeptide and further comprises an active promoter for expressing the polypeptide.
  • the promoter is selected from the group consisting of constitutive and inducible promoters.
  • the nucleotide sequence is a "sense” sequence; in another, the nucleotide sequence is an "antisense” sequence.
  • the present invention further contemplates the use of a composition comprising an adenovirus-derived protein and a therapeutic nucleotide sequence in the manufacture of a medicament for specifically targeting epithelial cells and delivering an effective amount of the therapeutic nucleotide sequence to the cells.
  • the invention contemplates the use of a compound comprising a therapeutic nucleotide sequence operatively linked to an adenovirus-derived protein in the manufacture of a medicament for specifically targeting epithelial cells and delivering an effective amount of the therapeutic nucleotide sequence to the cells.
  • the adenovirus-derived protein is selected from the group consisting of penton base and penton complex. In other variations, it will be appreciated that any combination of the preceding elements may also be efficacious for the uses as described herein.
  • the invention further contemplates methods of making a medicament useful in specifically targeting and delivering a therapeutic nucleotide sequence to mammalian cells, comprising operatively linking an adenovirus-derived amino acid residue sequence and the nucleotide sequence and incorporating the operatively linked sequences into a liposome.
  • the amino acid residue sequence comprises penton base or penton complex.
  • Other variations include combinations of elements as described hereinabove.
  • the present invention further contemplates an article of manufacture comprising packaging material and a composition effective for targeting epithelial cells and delivering a therapeutic nucleotide sequence to the cells, wherein the composition comprises an adenovirus-derived protein and a therapeutic nucleotide sequence, and wherein the packaging material comprises a label which indicates that the composition can be used for targeting epithelial cells and delivering a therapeutic nucleotide sequence to the cells.
  • the invention contemplates a composition useful in specifically targeting non-epithelial cells and delivering a therapeutic nucleotide sequence to the cells, comprising an adenovirus-derived protein-ligand conjugate and the nucleotide sequence.
  • the invention contemplates a composition designed to specifically target epithelial cells and deliver a therapeutic nucleotide sequence to the cells, comprising a protein or polypeptide including an RGD amino acid residue sequence, and the nucleotide sequence.
  • the composition is designed to specifically target endothelial cells.
  • the composition is designed to target malignant or tumor cells.
  • the adenovirus-derived protein is selected from the group consisting of penton base and penton complex.
  • the composition is immunotherapeutic.
  • the composition is designed to target cells expressing integrins, including, in various embodiments, receptors for vitronectin, fibronectin, collagen, la inin, thrombospondin, fibrinogen, and von Willebrandt' s factor.
  • the composition is designed to specifically target cells expressing a vitronectin receptor.
  • the composition is designed to specifically target cells expressing an ⁇ or an a v ⁇ > s receptor.
  • the therapeutic nucleotide sequence is operatively linked to the adenovirus-derived protein.
  • the therapeutic nucleotide sequence and the adenovirus-derived protein are contained within a liposome.
  • the nucleotide sequence encodes a polypeptide and further comprises an active promoter for expressing the polypeptide.
  • the promoter is selected from the group consisting of constitutive and inducible promoters.
  • the ligand may comprise an antibody, or may comprise an attachment sequence for a receptor.
  • the present invention further contemplates a method of specifically targeting and delivering a therapeutic nucleotide sequence into mammalian cells, comprising administration of a composition comprising an adenovirus-derived protein and the nucleotide sequence.
  • the adenovirus-derived protein is selected from the group consisting of penton base and penton complex.
  • the adenovirus-derived protein is recombinant; in others, the adenovirus-derived protein is conventionally-purified.
  • the invention further contemplates that the adenovirus-derived protein is derived from adenovirus type 2.
  • the therapeutic nucleotide sequence is operatively linked to the adenovirus- derived protein.
  • the therapeutic nucleotide sequence and the adenovirus- derived protein are contained within a liposome.
  • compositions of the present invention may be administered in various forms and by various means, including administration as an aerosol spray, intraperitoneally, via surgical implantation or injection, or via perfusion.
  • a composition according to the present invention is administered in a dosage range of 1 ⁇ g/ml to 1 mg/ml of active ingredient.
  • Figure 1 illustrates a schematic representation of an adenovirus. The mobilities and relative amounts of each protein after electrophoresis of the dissociated virus on a sodium dodecyl sulfate-containing polyacrylamide gel are shown on the right. The position of each polypeptide in the virion is designated; however, the configuration of the DNA does not imply the actual structure within the core.
  • the Roman numerals refer to a polypeptide designation described by Maizel, et al. (Virology 36: 126-136 (1968)).
  • the hexon (II), penton base (III), fiber (IV) , and hexon-associated proteins (Ilia, VI, VIII, and IX) are subunits of the capsid.
  • the core contains proteins V, VII, and u, as well as the 55 kD terminal protein covalently linked at each of the 5' ends of the linear DNA.
  • the two molecules of TP per virion are too few to be demonstrated by the Coomassie stain of the viral polypeptides, and u probably is one of the polypeptides found in the region of the gel containing proteins X to XII. (See Persson, et al. , Curr. Top. Microbiol. Immunol. 97: 157-203 (1982).)
  • Figure 2 illustrates a diagram of the adenovirus particle.
  • Fig. 2A is a diagram illustrating the adenovirus particle, showing the location of major protein sub-assemblies. The nine hexons drawn as a group and the "peripentonal" hexons are trimers of the same polypeptide. They are distinguished only by their location in the structure. (See Burnett, in McPherson, et al. (eds.), Biological Macromolecules and Assemblies. Vol. I, "The Viruses", Wiley, NY, pp. 337-385 (1984).)
  • Fig. 2B illustrates a cross-section, and shows the probable location of the principal polypeptide components and the viral DNA.
  • PL refers to poly L-lysine and pRSVLuc is a reporter plasmid containing a luciferase gene.
  • the samples identified by numbers 1-5 contained the following: (1) transferrin/PL, but no pRSVLuc; (2) pRSVLuc + transferrin/PL; (3) pRSVLuc + transferrin/PL + penton base + fiber; (4) pRSVLuc + transferrin/PL + penton base; (5) pRSVLuc + transferrin/PL + fiber.
  • Amino Acid Residue An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are preferably in the "L” isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxy-terminus.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of
  • DNA Homolog A nucleic acid having a preselected conserved nucleotide sequence and a sequence encoding a preferred polypeptide according to the present invention.
  • Downstream Further along a DNA sequence in the direction of sequence transcription or read out, that is traveling in a 3'- to 5' -direction along the non-coding strand of the DNA or 5'- to 3'-direction along the RNA transcript.
  • Expression The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
  • Gene A nucleic acid whose nucleotide sequence encodes an RNA or polypeptide.
  • a gene can be either RNA or DNA. Genes may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
  • Hybridization The pairing of substantially complementary nucleotide sequences (strands of nucleic acid) to form a duplex or heteroduplex by the establishment of hydrogen bonds between complementary base pairs. It is a specific, i.e. non-random, interaction between two complementary polynucleotides that can be competitively inhibited.
  • Ligand A molecule that contains a structural portion that is bound by specific interaction with a particular receptor protein.
  • Nucleotide A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate group, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glyco ⁇ idic carbon (1' carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose, it is referred to as a nucleotide.
  • a sequence of operatively linked nucleotides is typically referred to herein as a "base sequence” or “nucleotide sequence”, and their grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5' -terminus to 3'-terminus.
  • Nucleotide Analog A purine or pyrimidine nucleotide that differs structurally from A, T, G, C, or U, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
  • Oligonucleotide or Polynucleotide A polymer of single or double stranded nucleotides.
  • oligonucleotide and its grammatical equivalents will include the full range of nucleic acids.
  • An oligonucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art.
  • Penton The terms “penton” or “penton complex” are preferentially used herein to designate a complex of penton base and fiber. The term “penton” may also be used to indicate penton base, as well as penton complex. The meaning of the term “penton” alone should be clear from the context within which it is used.
  • Plasmid An autonomous self-replicating extrachromosomal circular DNA.
  • Polypeptide and Peptide are used interchangeably herein to designate a series of no more than about 50 amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • Protein As used herein, this term designates a linear series of greater than 50 amino acid residues connected one to the other as in a polypeptide.
  • Receptor and receptor protein are terms used herein to indicate a biologically active proteinaceous molecule that specifically binds to (or with) other molecules.
  • Recombinant DNA (rDNA) molecule A DNA molecule produced by operatively linking a nucleic acid sequence, such as a gene, to a DNA molecule sequence of the present invention.
  • a recombinant DNA molecule may be a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.
  • rDNAs not having a common biological origin, i.e., evolutionarily different, are said to be "heterologous” .
  • Therapeutic Nucleotide Sequence As described and claimed- herein, such a sequence includes DNA and RNA sequences encoding an RNA or polypeptide.
  • Such sequences may be "native" or naturally-derived sequences; they may also be recombinantly-derived sequences.
  • Therapeutic nucleotide sequences therefore include antisense sequences or nucleotide sequences which may be transcribed into antisense sequences.
  • Therapeutic nucleotide sequences further comprise sequences which function to produce a desired effect in the cell or cell nucleus into which said therapeutic sequences are delivered.
  • a therapeutic nucleotide sequence may encode a functional protein intended for delivery into a cell which is unable to produce that functional protein.
  • Transformation The acquisition of new genetic markers by incorporation of added DNA in procaryotic cells.
  • Vector A polynucleotide molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • Cloning Vector Any plasmid or virus into which a foreign DNA may be inserted to be cloned.
  • Expression Vector Any plasmid or virus into which a foreign DNA may be inserted or expressed. Vectors capable of directing the expression of DNA segments (genes) encoding one or more proteins are referred to herein as "expression vectors". Also included are vectors which allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.
  • Leader or Signal Polypeptide A short length of amino acid sequence at the amino end of a protein, which carries or directs the protein through the inner membrane and so ensures its eventual secretion into the periplasmic space. The leader sequence peptide is commonly removed before the protein becomes active. Reading Frame: A particular sequence of contiguous nucleotide triplets (codons) employed in translation. The reading frame depends on the location of the translation initiation codon. B. Adenoviruses
  • adenovirus Since its discovery in 1953, adenovirus has served as a model for molecular biology and cell transformation.
  • the pentagonal capsomer (the penton) at the vertex of the adenovirus icosahedron consists of a fiber projection, linked by noncovalent bonds to the penton base, anchored in the capsid.
  • the adenovirus (Ad) particle is relatively complex and may be resolved into various substructures.
  • the outer shell is strikingly icosahedral in shape and, at first glance, appears to have a triangulation number of 25.
  • the structures at the fivefold positions (“pentons”) are different from the rest (“hexons”), however, and the hexons are chemically trimers rather than hexamers.
  • the structure really does not correspond to a simple sub-triangulated icosahedral design. (See, e.g., Fields, et al . , Virology. Vol. I, Raven Press, NY, pp.
  • Adenoviruses are nonenveloped, regular icosahedrons (20 triangular surfaces and 12 vertices) that are about 65-80 nm in diameter (about 1400 angstroms (A) ) .
  • a structure, called fiber, projects from each of the vertices. The length of the fiber varies with the adenovirus serotype.
  • the protein coat or capsid is composed of 252 subunits (capsomeres) , of which 240 are hexons and 12 are pentons. Each of the pentons contains a penton base on the surface of the capsid and a fiber projecting from the base, which is surrounded by five hexons. The name "penton" is derived from these geometric relationships.
  • All the other capsomeres are hexons, and they are so named because each is surrounded by six neighboring identical structures.
  • a group of nine hexons (the "ninemers") can be purified from each of the 20 triangular faces of the virion by gentle lysis of the particles with 10% pyridine.
  • the hexons and pentons are each derived from different viral polypeptides.
  • Recently, combined electron-microscopic and crystallographic approaches have produced detailed descriptions of hexon-hexon and hexon-penton base interactions that have helped clarify the relationship of the virus subunits.
  • the hexon capsomere was shown to contain two ⁇ barrel configurations that were very similar to structures contained on the surface of several picornaviruses
  • the virion contains fiber polypeptides modified by covalent addition of glucosamine and has a density of 1.34 g/cm 3 in CsCl.
  • the hexon capsomere is a trimer of three identical 110K polypeptides, conventionally denoted polypeptide II, held tightly together by noncovalent interactions.
  • the 240 hexon capsomeres in each virion therefore contain 720 identical polypeptides.
  • the high-resolution structure shows that each subunit contains two rather similar ⁇ -barrel domains, in which the course of the polypeptide chain is somewhat like that in an RVC (RNA virus capsid) fold. That is, in the hexon domains there are two sheets of four strands each, connected sequentially as in the framework of an RVC domain.
  • the shape of the domain is rather different; the strands run radially rather than tangentially to the shell, and the major loops are between strands D and E and strands F and G.
  • use of a similarly folded design may not imply any evolutionary or functional relationship.
  • the DE and FG loops from both domains project outward, interacting tightly with corresponding loops from the other two subunits in the trimer. The conformation of these loops therefore depends on trimer formation.
  • the loops bear the principal type-specific antigenic determinants. Assembly of the hexon from newly synthesized protein in vivo appears to require a factor, the "100K protein", which is also encoded by the virus. This protein does not form part of the final structure. Dissociation of adenovirus particles by various methods yields groups of nine hexons, as shown in Fig. 2A. They are derived from virions as indicated, and they include all but the peripentonal hexons. The groups of nine are held together by viral protein IX (see Fig. 2B) , which copurifies with these structures. Its location has been determined by scanning electron microscopy. (See, e.g., Fields, et al., Virology, Vol. I, Raven Press, NY, pp. 54-56 (1990) .)
  • Polypeptides VI (24 kd) , VIII (13 kd) , and IX (12 kd) are associated with the hexon after various isolation procedures. For example, after disruption of the virion with 10% pyridine, polypeptides VI and IX are isolated together with hexon ninemers. Polypeptides VI and VIII are synthesized as larger precursors and are cleaved during assembly. The positions of these proteins within the virion are shown in Fig. 1, and those polypeptides facing the outer surface of the particle are indicated. The location of polypeptides was obtained by surface labeling of virion with iodinated lactoperoxidase or by analysis of various degradation products of gently disrupted particles. The penton base (polypeptide III, 85 kd) is noncovalently attached to fiber (polypeptide IV, 62 kd) .
  • the adenovirus penton is a noncovalently associated complex of two proteins, the fiber and penton base.
  • the penton complex comprises three fiber monomers associated with one penton base.
  • the complex of penton base plus fiber polypeptides is called penton capsomere.
  • the outer tip of the penton fiber is thought to be the site of attachment to cellular receptors. The orientation of the fiber polypeptide has been determined.
  • the fiber is an elongated protein (180,000 kDa) which exists as a trimer of three identical polypeptides of 582 amino acids in length (62 kDa) .
  • the N-terminus of the fiber mediates binding to the penton base while the C-terminus is involved in initial binding of the virus to cellular receptors.
  • Cellular receptors for the fiber have not yet been identified.
  • Adenovirus attachment to a putative host cell receptor is mediated by the fiber protein as demonstrated by the ability of soluble fiber to completely block virus attachment and infection.
  • the elongated portion of the fiber (shaft) is comprised of 22 repeats of 15 amino acids each (adenovirus type 2) which form amphipathic B sheet structure.
  • the adenovirus penton base is a pentameter comprised of 5 identical subunits of 571 amino acids which are noncovalently associated to form a ring-shaped complex of approximately 200 X 90 A. There is a 98% sequence identity in the penton base within the same subgroup of adenoviruses while there is only 69% identity in the fiber.
  • the penton base contains an arginine-glycine-aspartic acid-containing (RGD-containing) peptide sequence (i.e., HAIRGDTFA (SEQ ID NO 1) , residues 340-342 within residues 337-345) which are now shown to be involved in the attachment of the virus to integrin receptors.
  • adenovirus which is a nonenveloped DNA virus, utilizes separate proteins for attachment and entry in a manner similar to enveloped human viruses.
  • Adenoviruses attach to host-cell receptors via the penton fiber glycoprotein and enter cells through the process of receptor-mediated endocytosis mediated by the penton base.
  • the penton fiber projects from the vertices of the virion capsid. It has a small distal knob that contains the cell attachment site. It has been estimated that there are 10 4 virion binding sites per cell. There are a significantly larger number of cellular binding sites for the isolated penton fiber protein, indicating that the intact virion occupies several receptor sites.
  • Antibodies directed against the penton fiber neutralize viral infectivity.
  • Affinity columns made with the adenovirus penton capsomer have been used to isolate and partially purify the receptor for adenovirus on KB cells.
  • Three proteins of molecular mass 34, 42, and 78 kd have been isolated using this technique. These proteins have been presumed to comprise different forms of the same molecule or polypeptides which together form a part of a receptor complex.
  • the core of adenovirions contains DNA (about 30 kb) and two basic proteins (V and VII) .
  • the arginine-rich protein VII is present in about 1070 copies/particle, and it can neutralize about 50% of the DNA phosphates. Isolated cores are compact particles, but without any very striking substructures.
  • Adenovirus DNA is about 23.85 X 10 6 daltons for adenovirus type 2 (Ad 2) and varies slightly in size, depending on serotype.
  • Ad DNA is approximately 11 micromolar ( ⁇ m) in length and has the unusual feature of a virus-encoded 55 kd terminal polypeptide (TP) covalently linked to dCMP at each 5' end of the linear genome. Extensive proteolysis does not remove all the amino acids from the 5' terminal dCMP; however, the protein-DNA bond involving a serine hydroxyl group is alkaline-labile in sodium hydroxide or piperidine. Because TP does not allow the covalently linked fragments of DNA to enter agarose gels during electrophoresis without detergents, the end fragments of DNA can easily be determined for new serotypes.
  • Ad DNA molecules released from virions with 4M guanidine can be seen as circles formed by noncovalent interactions between the terminal proteins at each end.
  • Adenovirus DNA has inverted terminal redundancies and the 100- to 140-base-pair length of these repeats varies with the serotype. Therefore, denatured single strands of adenovirus DNA can form circles by base pairing at the ends of the DNA, and these "panhandle" structures may be important in DNA replication.
  • adenovirus-mediated gene therapy represents an improved method of DNA transfer into cells
  • a potential limitation of this approach is that adenovirus replication results in disruption of the host cell.
  • adenovirus also possesses oncogenic properties including the ability of one of its proteins to bind to tumor suppressor gene products.
  • the use of replication defective strains of adenovirus which render the virus unable to replicate in host cells is in principle more suitable for in vivo therapy; however, the potential of co-infection of epithelial cells with wild-type strains of virus resulting in transactivation of the recombinant virus may represent a significant safety concern for in vivo applications.
  • adenoviruses are capable of integrating their genome into host cell DNA allowing for long-term stable expression of the foreign gene.
  • Another undesirable aspect of using intact or replication-deficient adenovirus as a gene transfer means is that it is an oncogenic virus whose gene products are known to interfere with the function of host cell tumor suppressor proteins as well as immune recognition molecules, such as the major histocompatibility complex (MHC) .
  • MHC major histocompatibility complex
  • pre-existing circulating antibodies to adenovirus may significantly reduce the efficiency of in vivo gene delivery.
  • kb kilobases
  • the present invention uses a coat protein subunit of adenovirus known as the penton which duplicates cell receptor binding and DNA delivery properties of intact adenovirus virions and thus represents an improved method for gene therapy as well as antisense-based antiviral therapy.
  • the penton is composed of a fiber protein which mediates receptor binding and the penton base (Boudin, et al. , Virology 116: 589-604 (1982)) which mediates various entry and also disruption of the endocytic vesicle following exposure to a low pH environment thus allowing virus entry into the cytoplasm (Seth, et al. , J. Biol. Chem. 260: 9598-9602 (1985); Seth, et al. , J. Biol. Chem. 259: 14350-14353 (1984)).
  • adenovirus-derived penton base and fiber proteins provides certain advantages for gene delivery.
  • the adenovirus penton complex likely possesses all of the functional properties required for gene therapy including binding to epithelial cell receptors and penetration of endocytic vesicles.
  • penton base and fiber proteins can be purified directly from adenovirus using conventional purification techniques as described in Example 2, large amounts of recombinant penton base and fiber can be produced in insect cells using baculovirus as well as from other various expression systems following amplification of genes encoding the penton base and fiber.
  • the adenovirus type 2-derived recombinant penton base and fiber are preferred for use in this invention, the fiber and penton base proteins purified by conventional methods from adenovirus as described in Example 2 are also contemplated for use in this invention.
  • the recombinant proteins can be amplified from the available human adenovirus serotypes from type 1 through 47 currently available from American Type Culture Collection (ATCC) , Rockville, MD. Both recombinant and conventionally purified penton base and fiber proteins are capable of assembling into the penton complex. Use of adenovirus penton also eliminates the safety concerns related to the introduction of intact adenovirus genes into human cells.
  • Recombinant or conventionally purified adenovirus type 2 penton as well as the noncomplexed penton base and fiber proteins used independently are used in this invention to facilitate specific targeting and delivery of foreign genes into epithelial cells.
  • DNA or antisense-based plasmids are conjugated directly to a region of the penton such that its functional properties are not altered.
  • the penton complex, penton base or fiber is incorporated into liposomes which contain the foreign gene or antisense oligonucleotides.
  • the invention also uses plasmid-based vectors containing the gene of interest under the control of strong constitutive or inducible promoters and/or enhancer elements as described in Example 5 to obtain higher levels of gene expression than that obtained by incorporating the target gene into the adenovirus genome.
  • adenovirus penton will be used to target and deliver genes into non-epithelial cells by incorporating the attachment sequence for other receptors such as CD4 on T cells onto the fiber protein by recombinant DNA techniques, thus producing a chimeric molecule as described in Example 5.
  • the penton-based gene delivery system thus provides for increased flexibility in gene design to enable stable integration into proliferating and nonproliferating cell types.
  • this is contemplated by the incorporation of a retrovirus genome into the penton-based gene delivery system, the result of which allows for the stable integration of the exogenous gene into the recipient cell mediated by the retrovirus while allowing for delivery into nonproliferating cells mediated by the penton.
  • Gene transfer mediated by retroviruses outside this context is limited to proliferating cell types.
  • One application of this invention is gene therapy for common hereditary disorders involving respiratory epithelium such as alpha 1-antitrypsin deficiency resulting in emphysema and cystic fibrosis transmembrane conductance regulator (CFTR) deficiency resulting in cystic fibrosis.
  • a second application is delivery of antiviral agents such as antisense compounds or ribozymes to epithelial cells as described in Example 5.
  • the adenovirus penton may also be useful in the enhanced delivery of immunotoxins for the treatment of various cancers.
  • PCR Amplification of Penton Base and Fiber Proteins it is possible to synthesize useful polypeptide-encoding nucleotide sequences which may then be operatively linked to a vector and used to transform an appropriate cell and expressed therein.
  • PCR polymerase chain reaction
  • primer extension preferably by primer extension in a polymerase chain reaction (PCR) format.
  • PCR primer extension preferably by primer extension in a polymerase chain reaction (PCR) format.
  • the first primer becomes part of the nonsense (minus or complementary) strand and hybridizes to a nucleotide sequence conserved among the preferred gene's plus (or coding) strands. To produce coding DNA homologs, first primers are therefore chosen to hybridize to (i.e. be complementary to) conserved regions within the gene(s) of choice.
  • Second primers become part of the coding (plus) strand and hybridize to a nucleotide sequence conserved among minus strands.
  • second primers are therefore chosen to hybridize with a conserved nucleotide sequence at the 5' end of the coding gene such as in that area coding for the leader or first framework region.
  • conserved 5' nucleotide sequence of the second primer can be complementary to a sequence exogenously added using terminal deoxynucleotidyl transferase as described by Loh et al. , Science 243: 217-220 (1989) .
  • One or both of the first and second primers can contain a nucleotide sequence defining an endonuclease recognition site (restriction site) .
  • the site can be heterologous to the gene being amplified and typically appears at or near the 5' end of the primer.
  • the first primer of a PCR primer pair is sometimes referred to herein as the "sense primer” because it hybridizes to the coding or sense strand of a nucleic acid.
  • the second primer of a PCR primer pair is sometimes referred to herein as the "antisense primer” because it hybridizes to a non-coding or antisense strand of a nucleic acid, i.e., a strand complementary to a coding strand.
  • a plurality of first primers and/or a plurality of second primers can be used in each amplification, e.g., one species of first primer can be paired with a number of different second primers to form several different primer pairs. Alternatively, an individual pair of first and second primers can be used.
  • Primers are also referred to as being either 5' or 3' primers indicating the ends or region of the DNA to which the primers hybridize.
  • the 5' and 3' primers are respectively the antisense and sense primers.
  • the restriction site-defining portion is typically located in a 5' -terminal non-priming portion of the primer.
  • the restriction site defined by the first primer is typically chosen to be one recognized by a restriction enzyme that does not recognize the restriction site defined by the second primer, the objective being to produce a DNA molecule having cohesive termini that are non-complementary to each other and thus allow directional insertion into a vector.
  • each primer works in combination with a second primer to amplify a target nucleic acid sequence.
  • the choice of PCR primer pairs for use in PCR is governed by various considerations, as discussed herein. That is, the primers have a nucleotide sequence that is complementary to a sequence conserved in the gene of choice. Useful priming sequences are disclosed hereinafter.
  • the strategy used for cloning the selected genes will depend, as is well known in the art, on the type, complexity, and purity of the nucleic acids making up the various genes. Other factors include whether or not the genes are to be amplified and/or mutagenized.
  • the exemplary genes are comprised of polynucleotide coding strands, such as mRNA and/or the sense strand of genomic DNA. If the polynucleotide sequence is in the form of double stranded genomic DNA, it is usually first denatured, typically by melting, into single strands. A gene sequence is subjected to a PCR reaction by treating (contacting) the sequence with a PCR primer pair, each member of the pair having a preselected nucleotide sequence.
  • the PCR primer pair is capable of initiating primer extension reactions by hybridizing to nucleotide sequences, preferably at least about 10 nucleotides in length and more preferably at least about 20 nucleotides in length, conserved within the gene sequence.
  • the PCR reaction is performed by mixing the PCR primer pair, preferably a predetermined amount thereof, with the nucleic acids of the selected gene or DNA nucleotide sequence, preferably a predetermined amount thereof, in a PCR buffer to form a PCR reaction admixture.
  • the admixture is maintained under polynucleotide synthesizing conditions for a time period, which is typically predetermined, sufficient for the formation of a PCR reaction product, thereby producing a plurality of different polypeptide-encoding DNA homologs.
  • the PCR reaction is performed using any suitable method. Generally it occurs in a buffered aqueous solution, i.e., a PCR buffer, preferably at a pH of 7-9, most preferably about 8.
  • a molar excess for genomic nucleic acid, usually about 10 6 :l primer:template
  • a large molar excess is preferred to improve the efficiency of the process.
  • the PCR buffer also preferably contains the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP and a polymerase, typically thermostable, all in adequate amounts for primer extension (polynucleotide synthesis) reaction.
  • the resulting solution (PCR admixture) is heated to about 90°C - 100°C for about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period the solution is allowed to cool to 54°C, which is preferable for primer hybridization.
  • the synthesis reaction may occur at room temperature up to a temperature above which the polymerase (inducing agent) no longer functions efficiently.
  • An exemplary PCR buffer comprises the following: 50 mM KCl; 10 mM Tris-HCl at pH 8.3; 1.5 M MgCl 2 ; 0.001% (wt/vol) gelatin, 200 ⁇ M dATP; 200 ⁇ M dTTP; 200 ⁇ M dCTP; 200 ⁇ M dGTP; and 2.5 units Thermus aguaticus DNA polymerase I (U.S. Patent No. 4,889,818) per 100 microliters of buffer.
  • PCR for amplifying the recombinant penton base and fiber proteins of this invention was performed as described in Example 1.
  • the inducing agent may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase, and other enzymes, including heat-stable enzymes, which will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be inducing agents, however, which initiate synthesis at the 5' end and proceed in the above direction, using the same process as described above.
  • the inducing agent also may be a compound or system which will function to accomplish the synthesis of RNA primer extension products, including enzymes.
  • the inducing agent may be a DNA-dependent RNA polymerase such as T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. These polymerases produce a complementary RNA polynucleotide. The high turn-over rate of the RNA polymerase amplifies the starting polynucleotide as has been described by Chamberlin et al. , The Enzymes. ed. P. Boyer, PP. 87-108, Academic Press, New York
  • T7 RNA polymerase Another advantage of T7 RNA polymerase is that mutations can be introduced into the polynucleotide synthesis by replacing a portion of cDNA with one or more mutagenic oligodeoxynucleotides
  • the newly synthesized strand and its complementary nucleic acid strand form a double-stranded molecule which can be used in the succeeding steps of the process.
  • the DNA molecules are typically further amplified. While the DNA molecules can be amplified by classic techniques such as incorporation into an autonomously replicating vector, it is preferred to first amplify the molecules by subjecting them to a polymerase chain reaction (PCR) prior to inserting them into a vector.
  • PCR is typically carried out by thermocycling i.e., repeatedly increasing and decreasing the temperature of a PCR reaction admixture within a temperature range whose lower limit is about 10°C to about 40°C and whose upper limit is about 90°C to about 100°C.
  • the preferred amplification procedure was performed as described in Example 1.
  • the increasing and decreasing can be continuous, but is preferably phasic with time periods of relative temperature stability at each of temperatures favoring polynucleotide synthesis, denaturation and hybridization.
  • PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in several texts including "PCR Technology: Principles and Applications for DNA Amplification", H. Erlich, ed. , Stockton Press, New York (1989) ; and "PCR Protocols: A Guide to Methods and Applications", Innis et al. , eds . , Academic Press, San Diego, California (1990) .
  • Various preferred methods and primers used herein are described hereinafter and are also described in Nilsson, et al., Cell 58: 707 (1989) , Enni ⁇ , et al .
  • primers are preferred for amplification of penton base and fiber cDNA from adenovirus type 2 DNA, preferably in separate reactions.
  • the adenoviral template DNA is obtained as described in Example 1. Resulting cDNAs may then be cloned and sequenced as described herein. These primers are appropriate for use in amplifying all known and presently unknown types of adenovirus penton base and fiber.
  • the 5' and 3' primers used to amplify the penton base were, respectively, 5' -TTTCTAGAAGTATGCAGCGCGCG-3 ' (SEQ ID NO 2) and 5' -TTTCTAGATCAAAAAGTGCGGCT-3' (SEQ ID NO 3) .
  • the 5' and 3' primers used to amplify the fiber were, respectively, 5' -AAAGGATCCAGCTGATGAAACGCGCCA-3 ' (SEQ ID NO 4) and 5' -TTTGGTACCAGCTGTTATTCCTGGGCA-3' (SEQ ID NO 5) .
  • the restriction endonuclease cloning sites indicated by the underlined nucleotides are described in Example 1.
  • only one pair of first and second primers is used per amplification reaction.
  • the amplification reaction products obtained from a plurality of different amplifications, each using a plurality of different primer pairs, are then combined.
  • the present invention also contemplates DNA homolog production via co-amplification (using two pairs of primers) , and multiplex amplification (using up to about 8, 9 or 10 primer pairs) .
  • Expression of recombinant penton base and fiber proteins of this invention is accomplished through the use of expression vectors into which the PCR amplified penton base or fiber sequences described above have been inserted.
  • the expression vectors may be constructed utilizing any of the well-known vector construction techniques. Those techniques, however, are modified to the extent that the translatable nucleotide sequence to be inserted into the genome of the host cell is flanked "upstream" of the sequence by an appropriate promoter and/or enhancer sequences.
  • the vector also contains a selectable marker. After expression, the product of the translatable nucleotide sequence may then be purified using antibodies against that sequence.
  • neomycin resistance A plasmid encoding neomycin resistance, such as phshsneo, phsneo, or pcopneo, may be included in each transfection such that a population of cells that express the gene(s) of choice may be ascertained by growing the transfectants in selection medium.
  • the translatable nucleotide sequence may be incorporated into a plasmid with an appropriate controllable transcriptional promoter, translational control sequences, and a polylinker to simplify insertion of the translatable nucleotide sequence in the correct orientation, and may be expressed in the host cells.
  • Various host cells include a eucaryotic insect cell, such as Spodoptera frugiperda, or a procaryotic cell, such as E. coli .
  • E. coli - - it is necessary to use not only strong promoters to generate large quantities of mRNA, but also ribosome binding sites to ensure that the mRNA is efficiently translated.
  • the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides upstream from the initiation codon (Shine et al . , Nature, 254:34 (1975)) .
  • the sequence, AGGAGGU which is called the Shine-Dalgarno (SD) sequence, is complementary to the 3' end of E. coli 16S mRNA.
  • Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors, including (1) the degree of complementarity between the SD sequence and 3' end of the 16S tRNA; and (2) the spacing and possibly the DNA sequence lying between the SD sequence and the AUG.
  • Roberts et al. PNAS USA 76: 760 (1979a) ; Roberts et al . , PNAS USA 76: 5596 (1979b) ; Guarente et al., Science 209: 1428 (1980) ; and Guarente et al. , Cell 20 : 543 ( 1980 ) .
  • Vectors for use in producing large quantities of the recombinant adenovirus-derived proteins of this invention are designed for the expression of proteins in bacteria, in mammalian cells or in insect cells.
  • the expression vectors are preferably utilized in conjunction with bacterial "host" cells adapted for the production of useful quantities of proteins or polypeptides.
  • Such vectors may include a procaryotic replicon i.e., a nucleotide sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • those embodiments that include a procaryotic replicon may also include a gene whose expression confers a selective advantage, such as drug resistance, to a bacterial host transformed therewith.
  • a selective advantage such as drug resistance
  • typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
  • Vectors typically also contain convenient restriction sites for insertion of translatable nucleotide sequences.
  • the procaryotic expression vectors also contain promoters which can be used in the microbial organism for expression of its own proteins. Those promoters most commonly used include the beta-lacta ase and lactose promoter systems and the tryptophan promoter system as described in the European Patent Application No. 0125023.
  • Exemplary procaryotic expression vectors include the plasmids pUC8, pUC9, pUC18, pBR322, and pBR329 available from BioRad Laboratories (Richmond, CA) , pPL and pKK223 available from Pharmacia (Piscataway, NJ) , and pBS and M13mpl9 (Stratagene, La Jolla, CA) .
  • exemplary vectors include pCMU (Nilsson, et al. , Cell 58: 707 (1989) ) .
  • Other appropriate vectors may also be synthesized, according "to known methods; for example, vectors pCMU/K b and pCMUII used in various applications herein are modifications of pCMUIV (Nilsson, et al. , supra) .
  • Mammalian expression vector systems are also contemplated for the expression of recombinant penton base and fiber proteins for use in this invention.
  • viral-derived promoters are most commonly used.
  • frequently used promoters include polyoma, adenovirus type 2, and Simian Virus 40 (SV40) .
  • SV40 Simian Virus 40
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 base pair sequence extending from the Hind III restriction site toward the Bgl I site located in the viral origin of replication.
  • adenovirus 2 is also contemplated.
  • Origins of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral sources such as polyoma and adenovirus or may be provided by the host cell chromosomal replication mechanism. The latter is sufficient for integration of the expression vector in the host cell chromosome.
  • the preferred expression vector system for use preparing large quantities of recombinant penton base and fiber proteins is the baculovirus expression vector system, the details of which are described in Example 1. It will be understood that this invention, although described herein in terms of a preferred embodiment, should not be construed as limited to the host cells, expression vectors and expression vectors systems exemplified. Other expression vector systems, well known to one of ordinary skill in the art and described by Kaufman et al. , In “Current Protocols in Molecular Biology", Ausubel et al. , ed ⁇ . , Unit 16, New York (1990) , are contemplated for preparing recombinant penton base and fiber proteins for use in this invention.
  • the expression vectors containing the nucleotide sequences that encode the preferred proteins are transfected into the recipient hosts cells described above.
  • the host cell can be either procaryotic or eucaryotic.
  • Bacterial cells are preferred procaryotic host cells and typically are a strain of E. coli such as the MC1061 or JM109 strains.
  • Preferred eucaryotic host cells include yeast, insect and mammalian cells, where the latter are those from mouse, rat, monkey or human fibroblastic cell lines.
  • Preferred eucaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61 and NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC ar CRL1658. Most preferred for the expression of the recombinant penton base and fiber proteins is an insect cell, which include Spodoptera frugiperda and Trichoplu ⁇ ia ni strains as described in Example 1. Transfection may be accomplished via numerous methods, depending on the type of vector used, including the calcium phosphate method, the
  • Successfully transformed cells i.e., cells that contain the penton base or fiber expression vectors
  • an appropriate immunological, functional or visual assay For example, with the baculovirus vector expression vector system transfected into host insect cells, the screening for successfully transfected cells is accomplished by visual inspection in a plaque assay for the presence or absence of occlusion bodies as described in Example 1. Wild-type viral plaques contain the occlusions whereas recombinant transfected viral plaques lack occlusions.
  • successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of a protein of this invention. For example, samples of a culture containing cells suspected of being transformed are harvested and assayed for the subject expressed protein of this invention using either monoclonal or polyclonal antibodies that are specific for the expressed protein, such as anti-penton base or anti-fiber antibodies.
  • Purification of the expressed recombinant penton base and fiber proteins is accomplished by a variety of techniques depending on the selected expression vector system.
  • the viral stock containing the recombinant vector is used to infect host insect cells as described in Example 1.
  • the constitutively expressed recombinant proteins are then purified by lysing cells with hypotonic buffer and collecting the intracellular proteins by centrifugation.
  • penton base is capable of saturable and specific binding to human epithelial cells.
  • a therapeutic nucleotide composition of the present invention comprises a nucleotide sequence encoding a therapeutic molecule as described in Section F below.
  • a therapeutic nucleotide composition may further comprise an enhancer element or a promoter located 5' to and controlling the expression of said therapeutic nucleotide sequence or gene.
  • the promoter is a DNA segment that contains a DNA sequence that controls the expression of a gene located 3' or downstream of the promoter.
  • the promoter is the DNA sequence to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene, typically located 3' of the promoter.
  • the subject therapeutic nucleotide composition consists of a nucleic acid molecule that comprises at least 2 different operatively linked DNA segments.
  • the DNA can be manipulated and amplified by PCR as described in Section C and by using the standard techniques described in Molecular Cloning: A Laboratory Manual, 2nd Edition. Maniatis et al. , eds. , Cold Spring Harbor, New York (1989) .
  • the sequence encoding the selected therapeutic composition and the promoter or enhancer are operatively linked to a vector DNA molecule capable of autonomous replication in a cell either in vivo or in vi tro .
  • a recombinant DNA molecule (rDNA) of the present invention is a hybrid DNA molecule comprising at least 2 nucleotide sequences not normally found together in nature.
  • the therapeutic nucleotide composition of the present invention is from about 20 base pairs to about 100,000 base pairs in length.
  • the nucleic acid molecule is from about 50 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid molecule is from about 50 base pairs to about 10,000 base pairs in length. Most preferred is a nucleic acid molecule from about 50 pairs to about 4,000 base pairs in length.
  • the therapeutic nucleotide can be a gene or gene fragment that encodes a protein or peptide that provides the desired therapeutic effect such as replacement of alpha 1-antitrypsin or cystic fibrosis transmembrane regulator protein and the like.
  • the therapeutic nucleotide can be a DNA or RNA oligonucleotide sequence that exhibits enzymatic therapeutic activity.
  • examples of the latter include antisense oligonucleotides that will inhibit the transcription of deleterious genes or ribozymes that act as site-specific ribonucleases for cleaving selected mutated gene sequences.
  • a therapeutic nucleotide sequence of the present invention may comprise a DNA construct capable of generating therapeutic nucleotide molecules, including ribozymes and antisense DNA, in high copy numbers in target cells, as described in published PCT application No. WO 92/06693 (the disclosure of which is incorporated herein by reference) .
  • a regulatable promoter is a promoter where the rate of RNA polymerase binding and initiation is modulated by external stimuli. Such stimuli include compositions light, heat, stress and the like. Inducible, suppressible and repressible promoters are regulatable promoters. Regulatable promoters may also include tissue specific promoters. Tissue specific promoters direct the expression of that gene to a specific cell type. Tissue specific promoters cause the gene located 3' of it to be expressed predominantly, if not exclusively in the specific cells where the promoter expressed its endogenous gene. Typically, it appears that if a tissue-specific promoter expresses the gene located 3' of it at all, then it is expressed appropriately in the correct cell types as has been reviewed by Palmiter et al. , Ann. Rev. Genet. 20: 465-499 (1986) .
  • tissue specific promoter When a tissue specific promoter controls the expression of a gene, that gene will be expressed in a small number of tissues or cell types rather than in substantially all tissues and cell types.
  • tissue specific promoters include the immunoglobulin promoter described by Brinster et al. , Nature 306: 332-336 (1983) and Storb et al. , Nature 310: 238-231 (1984) ; the elastase-I promoter described by Swift et al., Cell 38: 639-646 (1984); the globin promoter described by Townes et al. , Mol. Cell. Biol. 5: 1977-1983 (1985), and Magram et al. , Mol. Cell. Biol.
  • BTSH beta-thyroid stimulating hormone
  • MMTV mouse mammary tumor virus
  • NSE neuron-specific enolase
  • exogenous DNA into eucaryotic cells has become one of the most powerful tools of the molecular biologist.
  • exogenous encompasses any therapeutic composition of this invention which is administered by the therapeutic methods of this invention.
  • exogenous is also referred to as “foreign, nonnative, and the like".
  • the methods of this invention requires efficient delivery of the DNA into the nucleus of the recipient cell and subsequent identification of cells that are expressing the foreign DNA.
  • Engineered vectors such as plasmids or bacteriophages (phages) or other DNA sequences that are able to replicate in a host cell can be used to construct cells that act as factories to produce large amounts of specific viral proteins.
  • Recombinant plasmids will be used herein as exemplary vectors, also called cloning vehicles. See U.S. Patent No. 4,338,397, incorporated herein by reference.
  • Plasmids are extrachromosomal genetic elements found in a variety of bacterial species. They are typically double-stranded, closed, circular DNA molecules.
  • a widely-used plasmid is pBR322, a vector whose nucleotide sequence and endonuclease cleavage sites are well known. Nucleic acid production using plasmid or phage vectors has become very straightforward.
  • the plasmid or phage DNA is cleaved with a restriction endonuclease and joined in vivo to a foreign DNA of choice.
  • the resulting recombinant plasmid or phage is then introduced into a cell such as E. coli , and the cell so produced is induced to produce many copies of the engineered vector.
  • the produced foreign DNA is excised and placed into a second vector to produce or transcribe the protein or polypeptide encoded by the foreign gene.
  • Expression vectors contain sequences of DNA that are required for the transcription of cloned genes and the translation of their messenger RNA's (mRNA's) into proteins. Typically, such required sequences or control elements are: (1) a promoter that signals the starting point for transcription; (2) a terminator that signals the ending point of transcription; (3) an operator that regulates the promotor; (4) a ribosome binding site for the initial binding of the cells' protein synthesis machinery; and (5) start and stop codons that signal the beginning and ending of protein synthesis.
  • an expression vector should possess several additional properties. It should be relatively small and contain a strong promoter. The expression vector should carry one or more selectable markers to allow identification of transformants. It should also contain a recognition site for one or more restriction enzymes in regions of the vector that are not essential for expression.
  • transformed cells are cultured in the presence of radioactivity after immunoprecipitation. This approach has used
  • Staphylococcu ⁇ aureu ⁇ protein A selection of immune complexes (Kessler, J. Immunol. 115: 1617-1624 (1975) ) and the Western blotting procedure (Renart et al. , PNAS USA 76: 3116-3120 (1979)) to detect transformation-specific markers.
  • a vector of the present invention is a nucleic acid (preferably DNA) molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • a DNA segment e.g., gene or polynucleotide
  • one of the nucleotide segments to be operatively linked to vector sequences encodes at least a portion of a therapeutic molecule.
  • the entire peptide-coding sequence of the therapeutic gene is inserted into the vector and expressed; however, it is also feasible to construct a vector which also includes some non-coding sequences as well.
  • the non-coding sequences are excluded.
  • nucleotide sequence for a soluble form of a polypeptide may be utilized.
  • Another preferred vector includes a nucleotide sequence encoding at least a portion of a therapeutic nucleotide sequence operatively linked to the vector for expression.
  • vector refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked.
  • Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the nucleotide (DNA) segments to which they are operatively linked.
  • operatively linked means the sequences or segments have been covalently joined into one piece of DNA, whether in single or double stranded form.
  • a vector is utilized for the production of therapeutic proteins or polypeptides useful in the present invention.
  • Such vectors are preferably utilized in conjunction with eucaryotic cells.
  • Such vectors may include a eucaryotic replicon i.e., a nucleotide sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally or stably in a eucaryotic recipient cell.
  • replicons are well known in the art.
  • those embodiments that include a eucaryotic replicon may also include a gene whose expression confers a selective advantage, such as drug resistance.
  • Vectors typically also contain convenient restriction sites for insertion of translatable nucleotide sequences.
  • Exemplary vectors include the Chinese hamster ovary cell expression vectors, vaccinia virus expression system, vaccinia virus/T7 RNA polymerase hybrid system and the like as described by Kaufman et al. , In “Current Protocols in Molecular Biology", Ausubel et al. , eds., Unit 16.12.5, New York (1990).
  • a sequence of nucleotides adapted for directional ligation is a region of the expression vector that (1) operatively links for replication and transport the upstream and downstream nucleotide sequences and (2) provides a site or means for directional ligation of a nucleotide sequence into the vector.
  • a directional polylinker is a sequence of nucleotides that defines two or more restriction endonuclease recognition sequences, or restriction sites. Upon restriction cleavage, the two sites yield cohesive termini to which a translatable nucleotide sequence can be ligated to the expression vector.
  • the two restriction sites provide, upon restriction cleavage, cohesive termini that are non-complementary and thereby permit directional insertion of a translatable nucleotide sequence into the vector.
  • the directional ligation means is provided by nucleotides present in the upstream nucleotide sequence, downstream nucleotide sequence, or both.
  • the sequence of nucleotides adapted for directional ligation comprises a sequence of nucleotides that defines multiple directional cloning means. Where the sequence of nucleotides adapted for directional ligation defines numerous restriction sites, it is referred to as a multiple cloning site.
  • a translatable nucleotide sequence is a linear series of nucleotides that provide an uninterrupted series of at least 8 codons that encode a polypeptide in one reading frame.
  • the nucleotide sequence is a DNA sequence.
  • the vector itself may be of any suitable type, such as a viral vector (RNA or DNA) , naked straight-chain or circular DNA, or a vesicle or envelope containing the nucleic acid material and any polypeptides that are to be inserted into the cell.
  • RNA or DNA viral vector
  • naked straight-chain or circular DNA or a vesicle or envelope containing the nucleic acid material and any polypeptides that are to be inserted into the cell.
  • vesicles techniques for construction of lipid vesicles, such as liposomes described in Section E, are well known.
  • Such liposomes may be targeted to particular cells using other conventional techniques, such as providing an antibody or other specific binding molecule on the exterior of the liposome. See, e.g., A. Huang, et al., J. Biol. Chem. 255: 8015-8018 (1980).
  • Most useful vectors contain multiple elements including one or more of the following, depending on the nature of the recipient cell: an SV40 origin of replication for amplification to high copy number; an efficient promoter element for high-level transcription initiation; mRNA processing signals such as mRNA cleavage and polyadenylation sequences (and frequently, intervening sequences as well) ; polylinkers containing multiple restriction endonuclease sites for insertion of foreign DNA; selectable markers that can be used to select cells that have stably integrated the plasmid DNA; and plasmid replication control sequences to permit propagation in bacterial cells.
  • many vectors also contain an inducible expression system that is regulated by an external stimulus.
  • Sequences from a number of promoters that are required for induced transcription have been identified and engineered into expression vectors to obtain inducible expression.
  • Several useful inducible vectors have been based on induction by ⁇ -interferon, heat-shock, heavy metal ions, and steroids (e.g. glucocorticoids) .
  • steroids e.g. glucocorticoids
  • Other promoters contemplated for use in this invention are described in Example 5.
  • a preferred vector in which therapeutic nucleotide compositions of this invention are present is a plasmid; more preferably, it is a high-copy-number plasmid.
  • the vector contain an inducible promoter sequence, as inducible promoters tend to limit selection pressure against cells into which such vectors (which are often constructed to carry non-native or chimeric nucleotide sequences) have been introduced by the adenovirus-derived proteins of this invention. It is also preferable that the vector of choice be best suited for expression in the preselected recipient cell type depending on the nature of the gene replacement therapy.
  • a tissue containing a therapeutic nucleotide sequence of the present invention may also be produced by directly introducing the vector containing the sequence into an animal or by linking the therapeutic oligonucleotide directly to the adenovirus-derived proteins.
  • Direct vector delivery in vivo may be accomplished by transducing the desired cells and tissues with viral vectors or other physical gene transfer vehicles.
  • Other physical agents including naked plasmids, cloned genes encapsulated in targetable liposomes (see Section E below) or in erythrocyte ghosts have been use to introduce genes, proteins, toxins and other agents directly into whole animals.
  • Direct injection of therapeutic nucleotide sequences is also a viable alternative for delivery of therapeutic sequences.
  • the injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene the rat livers.
  • Kaneda, et al. Science, 243:375 (1989), incorporated by reference herein.
  • a subject therapeutic sequence, penton base, fiber or penton complex, penton conjugates and compositions of the present invention is covalently linked to a carrier, such as a phospholipid. More preferably, the active ingredient is removably inserted into a liposome, i.e., incorporated
  • Therapeutic nucleotide sequences and compositions according to the present invention can be administered in a liposome (micelle) formulation which can be administered by application to mucous membranes of body cavities.
  • a liposome micelle
  • Liposomes are prepared by a variety of techniques well known to those skilled in the art to yield several different physical structures, ranging from the smallest unilamellar vesicles of approximately 20 to 50 nanometers in diameter up to multilamellar vesicles of tens of microns in diameter.
  • Gregoriadis ed.), Liposome Technology 1. CRC Press (1984) .
  • Therapeutic nucleotide sequences and compositions for nasal formulations are preferably hydrated with a lyophilized powder of multilamellar vesicles to form liposomes containing the sequences and compositions according to the present invention.
  • Therapeutic sequences, penton base, fiber or penton complex, penton conjugates and compositions of the present invention may also be incorporated into liposomal vesicles via reverse loading (see U.S. Pat. No. 5,104,661), or in the manner described for the incorporation of amphotericin B into lipid vesicles. (See, e.g., Lopez-Berenstein, et al. , J. Infect. Pis.
  • Delivery may also be accomplished using liposomes with enhanced circulation time (see U.S. Pat. No. 5,013,556).
  • Use of liposomes to deliver therapeutic nucleotide sequences according to the present invention may also be utilized to regulate expression of target sequences or to limit the proliferation of virus or retrovirus. (See, e.g., published PCT application No. WO 92/06192, incorporated herein by reference.)
  • the amount of active ingredient incorporated into liposomes may be about 0.1 ⁇ g active ingredient per mg lipid to about 1 mg per mg lipid.
  • the dosage amount of active ingredient administered in lipid encapsulated form is preferably about 0.1-lmg per mg lipid.
  • Direct vector delivery in vivo may be accomplished via liposomal delivery.
  • Various physical agents including therapeutic nucleotide sequences encapsulated in targetable liposomes have been use to introduce genes, proteins, toxins and other agents directly into whole animals. See, for example, the liposome-mediated gene delivery in vivo and expression of preproinsulin genes in recipient rats described by Nicolaou, et al. , PNAS USA 80:1068 (1983) and Soriano, et al., PNAS USA 80:7128 (1983) . All cited disclosures are incorporated herein by reference.
  • compositions of the present invention may contain a physiologically tolerable carrier together with one or more therapeutic nucleotide sequences of this invention, dissolved or dispersed therein as an active ingredient.
  • the composition is not immunogenic or otherwise able to cause undesirable side effects when administered to a mammal or human patient for therapeutic purposes.
  • the terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • the present invention comprises therapeutic compositions useful in the specific targeting of epithelial or non-epithelial cells as well as in delivering a therapeutic nucleotide sequence to those cells.
  • Therapeutic compositions designed to preferentially target to epithelial cells may comprise an adenovirus-derived protein and a therapeutic nucleotide sequence.
  • penton base or penton complex are preferred adenovirus-derived proteins useful in the presently-disclosed therapeutic compositions and methods.
  • Compositions designed to preferentially target non-epithelial cells may include an adenovirus-derived protein-ligand conjugate and a therapeutic nucleotide sequence.
  • useful ligands directed to specific receptors include the V3 loop of HIV gpl20 (CD4) ; transferrin (transferrin receptor) ; LDL (LDL receptors) ; and deglycosylated proteins (asialoglycoprotein receptor) .
  • Polypeptides having a sequence that includes an amino acid residue sequence selected from the group comprising -EDPGFFNVE- (SEQ ID NO 6) and -EDPGKQLYNVE- (SEQ ID NO 7) are capable of targeting receptors such as the CR2 receptor, and are thus useful in compositions disclosed herein.
  • Useful ligands also include antibodies and attachment sequences, as well as receptors themselves. Antibodies to cell receptor molecules such as integrins and the like, MHC Class I and Class II, asialoglycoprotein receptor, transferrin receptors, LDL receptors, CD4, and CR2 are but a few useful according to the present invention. It is also understood that the ligands typically bound by receptors, as well as analogs to those ligands, may be used as cellular targeting agents as disclosed herein. Exemplary and preferred nucleotide sequences encode an expressible peptide, polypeptide or protein, and may further include an active constitutive or inducible promoter sequence.
  • preferred therapeutic nucleotide sequences according to the present invention are capable of delivering HIV antisense nucleotides to latently-infected T cells via CD4.
  • delivery of Epstein-Barr Virus (EBV) EBNa-1 antisense nucleotides to B cells via CR2 is capable of effecting therapeutic results.
  • EBV Epstein-Barr Virus
  • compositions that contains active ingredients dissolved or dispersed therein are well understood in the art.
  • compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the preparation can also be emulsified, or formulated into suppositories, ointments, creams, dermal patches, or the like, depending on the desired route of administration.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof, including vegetable oils, propylene glycol, polyethylene glycol and benzyl alcohol (for injection or liquid preparations) ; and vaseline, vegetable oil, animal fat and polyethylene glycol (for externally applicable preparations) .
  • the composition can contain wetting or emulsifying agents, isotonic agents, dissolution promoting agents, stabilizers, colorants, antiseptic agents, soothing agents and the like additives (as usual auxiliary additives to pharmaceutical preparations) , pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • compositions of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art.
  • liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.
  • additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • a therapeutic composition typically contains an amount of a therapeutic nucleotide sequence of the present invention sufficient to deliver a therapeutically effective amount to the target tissue, typically an amount of at least 0.1 weight percent to about 90 weight percent of therapeutic nucleotide sequence per weight of total therapeutic composition.
  • a weight percent is a ratio by weight of therapeutic nucleotide sequence to total composition.
  • 0.1 weight percent is 0.1 grams of DNA segment per 100 grams of total composition.
  • the therapeutic nucleotide compositions comprising synthetic oligonucleotide sequences of the present invention can be prepared using any suitable method, such as, the phosphotriester or phosphodiester methods. See Narang et al., Meth. Enzymol. , 68:90, (1979); U.S. Patent No. 4,356,270; and Brown et al. , Meth. Enzvmol.. 68:109, (1979).
  • the synthesis of the family members can be conducted simultaneously in a single reaction vessel, or can be synthesized independently and later admixed in preselected molar ratios.
  • the nucleotide residues that are conserved at preselected positions of the sequence of the family member can be introduced in a chemical synthesis protocol simultaneously to the variants by the addition of a single preselected nucleotide precursor to the solid phase oligonucleotide reaction admixture when that position number of the oligonucleotide is being chemically added to the growing oligonucleotide polymer.
  • nucleotide residues to those positions in the sequence that vary can be introduced simultaneously by the addition of amounts, preferably equimolar amounts, of multiple preselected nucleotide precursors to the solid phase oligonucleotide reaction admixture during chemical synthesis.
  • amounts preferably equimolar amounts
  • nucleotide precursors for example, where all four possible natural nucleotides (A,T,G and C) are to be added at a preselected position, their precursors are added to the oligonucleotide synthesis reaction at that step to simultaneously form four variants.
  • Nucleotide bases other than the common four nucleotides (A,T,G or C) , or the RNA equivalent nucleotide uracil (U) , can be used in the present invention.
  • inosine (I) is capable of hybridizing with A, T and G, but not C.
  • the preselected therapeutic nucleotide composition is designed to contain oligonucleotides that can hybridize to four sequences that vary at one position, several different oligonucleotide structures are contemplated.
  • the composition can contain four members, where a preselected position contains A,T,G or C.
  • the composition can contain two members, where a preselected position contains I or C, and has the capacity the hybridize at that position to all four possible common nucleotides.
  • other nucleotides may be included at the preselected position that have the capacity to hybridize in a non-destabilizing manner with more than one of the common nucleotides in a manner similar to inosine.
  • proteins derived from adenovirus are capable of delivering a therapeutic nucleotide sequence to a specific cell or tissue, thereby expanding and enhancing treatment options available in numerous conditions in which more conventional therapies are of limited efficacy.
  • the therapeutic nucleotide sequences described herein and compositions including same have a number of uses, and may be used in vi tro or in vivo.
  • the compositions may be used prophylactically or therapeutically in vivo to disrupt HIV infection and mechanisms of action by inhibiting gene expression or activation, via delivery of antisense HIV sequences or ribozymes to T cells or monocytes.
  • Other useful therapeutic nucleotide sequences include antisense nucleotide sequences complementary to EBV EBNa-1 gene. Use of such therapeutic sequences may remediate or prevent latent infection of B cells with EBV. (As discussed in Example 5 below, targeting and delivery may be accomplished via the use of various ligands, receptors, and other appropriate targeting agents.)
  • the method comprises, in one embodiment, contacting human cells infected with EBV or HIV with a therapeutically effective amount of a pharmaceutically acceptable composition comprising a therapeutic nucleotide sequence of this invention.
  • the contacting involves introducing the therapeutic nucleotide sequence composition into cells having an EBV or HIV-mediated infection.
  • the present invention also contemplates methods for determining the efficacy of the within-disclosed therapeutic compositions and methods.
  • One such method for confirming efficacy utilizes the human/SCID (severe combined immunodeficient) mouse model of EBV- induced LPD (lymphoproliferative disease) to ascertain whether EBV-antisense therapeutic nucleotide sequences block tumor formation.
  • SCID/hu chimeric mice resulting from SCID mice reconstituted with human lymphocytes from EBV positive donors develop aggressive human B-cell tumors containing viral DNA, or fatal LPD of human B-cell origin containing EBV DNA.
  • SCID mice are injected with lymphocytes from peripheral blood (PBL) or palatine tonsils (lymph nodes, LN) from EBV-seronegative individuals.
  • the SCID/hu mice are then infected with EBV about 7 days thereafter.
  • Administration of therapeutic nucleotide sequences may proceed prior to, concurrent with, or shortly after the EBV infection. Since EBV-infected SCID/hu mice tend to develop clinical signs of tumor 1-2 months after EBV infection, the mice are monitored over this period of time to ascertain the effect of the therapeutic nucleotide sequences and compositions on the course of the infection and tumor (or LPD) progression.
  • SCID/hu mice may be monitored to ascertain whether circulating cells with human and EBV DNA are present, as these cells tend to appear before the appearance of overt tumors (Pisa, et al. , Blood 79: 173-179 (1992) . ) '
  • Ad2 human adenovirus type 2
  • PCR polymerase chain reaction
  • HeLa cells American Type Culture Collection, Rockville, MD (ATCC accession number CCL 2)
  • Eagle's MEM with non-essential amino acids and 90% Earle's BSS containing 10% fetal bovine serum
  • Ad2 obtained from ATCC having ATCC accession number VR-846 having a multiplicity of infection (MOD of 10.
  • Virus infected cells were then harvested 2-3 days later by pelleting by centrifugation the cells at 10,000 rpm for 10 minutes at 4°C. The harvested cells were freeze-thawed 5 times to release intracellular particles and then the cell debris was removed by centrifugation.
  • CsCl 2 cesium chloride
  • the 5' and 3' primers used to amplify the penton base were, respectively, 5' -TTTC AGAAGTATGCAGCGCGCG-3' (SEQ ID NO 2) and 5' -TTTCTAGATCAAAAAGTGCGGCT-3' (SEQ ID NO 3).
  • the underlined nucleotides indicate the nucleotide sequence for incorporating the restriction enzyme site Xba I into the amplified penton base nucleotide sequence for subsequent cloning into the compatible Nhe I restriction site in the 14 kilobase (kb) baculovirus expression vector, pBlueBac II (Invitrogen, San Diego, CA) .
  • the 5' and 3' primers used to amplify the fiber were, respectively, 5' -AAAGGATCCAGCTGATGAAACGCGCCA-3' (SEQ ID NO 4) and 5' -TTTGGTACCAGCTGTTATTCCTGGGCA-3' (SEQ ID NO 5) .
  • the first six underlined nucleotides (GGATCC) in the 5' oligonucleotide (SEQ ID NO 4) indicate the nucleotide sequence for incorporating the restriction enzyme site Bam HI into the amplified fiber nucleotide sequence.
  • the second six underlined nucleotides (CAGCTG) with a C nucleotide overlap in the 5' oligonucleotide indicate the nucleotide sequence for incorporating the restriction enzyme site Pvu II into the amplified fiber nucleotide sequence.
  • the 5' end of the 3' oligonucleotide (SEQ ID NO 5) contained a Kpn I (GGTACC) restriction site (the first six nucleotides) .
  • a Pvu II restriction site (CAGCTG) with a C nucleotide overlap was also incorporated into the 3 ' oligonucleotide as indicated by the underlined sequence.
  • the restriction sites identified herein were amplified into the 5' and 3' ends of the fiber PCR product for cloning the resultant into the pBlueBac vector as described below.
  • the penton base and fiber nucleotide sequences were separately amplified by PCR in reactions containing 85 microliters ( ⁇ l) water, 10 ⁇ l PCR 10X buffer (100 millimolar (mM) Tris-HCl at pH 8.0, 500 mM KC1, 15 mM MgCl 2 and 0.1% (w/v) gelatin) , 1 ⁇ l each of the 5' and 3' oligonucleotide primers listed above for a final concentration of 50 picomolar/ ⁇ l, 1 ⁇ l 100X dNTP's (final concentration of 200 micro olar ( ⁇ M) each) , 1 ⁇ l Thermu ⁇ aquaticu ⁇ (Taq) polymerase (Perkins-Elmer Corp., Norwalk, CT) , and 10 9 plaque forming units of purified viral DNA prepared above. Thirty amplification cycles were performed with one cycle consisting of 2 minutes at 72°C, 10 minutes at
  • the PCR product contained 1713 base pairs (bp) for encoding the penton base bordered by Xba I restriction site nucleotides.
  • the nucleotide sequence for the penton base gene of human adenovirus type 2 has been described by Neumann et al. , Gene
  • the fiber PCR product contained 1746 bp for encoding the fiber bordered by Bam HI/Pvu II and Pvu II/Kpn I restriction site nucleotides.
  • the nucleotide sequence for the fiber gene of human adenovirus type 2 has been described by Herisse et al., Nuc. Acids Res. 9:4023-4041 (1981) .
  • the resultant PCR products were purified and separately ligated into pBlueBac II baculovirus expression vector in the MaxBac ® purchased from Invitrogen (San Diego, CA) .
  • the penton base having
  • Xba I restriction sites on both the 5' and 3' ends was first digested with Xba I then ligated into the pBlueBac II vector previously digested with Nhe I to form a recombinant pBlueBac II penton base DNA-containing vector.
  • Nhe I and Xba I are compatible restriction sites thereby providing for ligation of the PCR product into the expression vector.
  • the fiber PCR product was first digested with Bam HI and Kpn I and directionally ligated into a similarly digested pBluescript vector (Stratagene, La Jolla, CA) .
  • the fiber nucleotide sequence was then removed from pBluescript with the blunt end cutter, Pvu II, after which Nhe I adapter oligonucleotides (Invitrogen) were ligated to the isolated fiber sequence.
  • the resultant fiber nucleotide sequence having Nhe I ends was then ligated into the Nhe I digested pBlueBac II vector to form a recombinant pBlueBac II fiber DNA-containing vector.
  • the baculovirus expression vector is a helper-independent recombinant virus vector system used for expressing genes from sources including bacteria, viruses, plants and mammals. Recombinant fusion or nonfusion proteins have been reported to be expressed at levels ranging from 1-500 mg/liter.
  • the pBlueBac II expression vector was derived from the pJVNhe I expression vector described by Vialard et al. , J. Virol. 64:37-50 (1990). As the pBlueBac II encodes ⁇ -galactosidase, the screening of recombinant baculovirus is simplified thereby not requiring cotransfection with a selectable marker-based approach.
  • the vector has a unique ⁇ he I restriction cloning site and the 5' polyhedrin mR ⁇ A leader sequence of pVL941 is also utilized.
  • the vector contains two promoters, the polyhedrin promoter of Autographa californica multiple nuclear polyhedrosis virus (AcM ⁇ PV) and the plO promoter, that are both activated very late in viral infection.
  • the gene of interest is cloned downstream of the polyhedrin promoter which then controls synthesis of the recombinant protein.
  • the vector also contains the lacZ gene positioned downstream of the plO promoter which directs the synthesis of ⁇ -galactosidase.
  • the recombinant virus is then plaqued; recombinant virus generates plaques that are blue and lack occlusion bodies.
  • Spodoptera frugiperda (Sf9) cells were used for transfecting the recombinant DNA expression vectors of this invention. After 6 days post-viral infection of the cultured cells, the culture plates were examined under a dissecting microscope to select plaques that are occlusion body negative. Wild-type plaques were distinguished by the presence of occlusion bodies. Following selection of occlusion body negative recombinant plaques, the recombinant virus was purified away from contaminating wild-type virus in order to obtain a pure recombinant virus containing either penton base or fiber containing recombinants.
  • the selected plaques are analyzed by PCR as described above to confirm the presence of the recombined Ad2 gene and selection of the desired recombinant virus.
  • the viruses were propagated to prepare large scale high titer virus stocks as described in the MaxBac ® TM manual provided by Invitrogen and with procedures well known to one of ordinary skill in the art. The virus titer was then determined prior to preparation of recombinant penton base and fiber. Trichoplu ⁇ ia ni (Bti-Tn 5B1-4) cells
  • Tn 5 cells were used to express large amounts of recombinant protein since they grow as adherent cell lines and produce recombinant proteins in amounts that surpasses that produced in Sf9 cells.
  • Tn 5 cells were used to express large amounts of recombinant protein since they grow as adherent cell lines and produce recombinant proteins in amounts that surpasses that produced in Sf9 cells.
  • Tn 5 cells were used to express large amounts of recombinant protein since they grow as adherent cell lines and produce recombinant proteins in amounts that surpasses that produced in Sf9 cells.
  • Tn 5 cells were used to express large amounts of recombinant protein since they grow as adherent cell lines and produce recombinant proteins in amounts that surpasses that produced in Sf9 cells.
  • Tn 5 cells were used to express large amounts of recombinant protein since they grow as adherent cell lines and produce recombinant proteins in amounts that surpasses that produced in Sf9 cells.
  • Tn 5 cells
  • Tn 5 cells were purchased from Invitrogen and maintained in tissue culture flasks in EX-CELL 400 insect cell medium (JRH Biosciences, Lenexa, KS) supplemented with 2% fetal bovine serum -(Irvine
  • the cultures were also adapted for growth in DEAE-based Dormacell 1.2 microcarrier-coated roller bottles (JRH Biosciences) for larger-scale recombinant protein production.
  • the microcarriers were added to PBS at 25 mg/ml and autoclaved.
  • Fifty ml of the microcarrier suspension was added to 850 cm 2 polystyrene roller bottles and allowed to attach by slowly rotating the roller bottle at 0.1 rpm.
  • the roller bottle was washed 3 times with 50 ml of sterile saline solution to remove unbound microcarriers.
  • the saline solution was then removed and a suspension of Tn 5 cells was added and allowed to attach and grow at 4 rpm.
  • the volume of cells used for the inoculation was always kept below 100 ml to allow the cells to quickly attach to the microcarriers and to prevent cell clumping. The volume was then adjusted with fresh medium after the cells had attached which was approximately 2-3 hours later.
  • Tn 5 cells were directly plated at densities of 0.75 X 10 4 to 5 X 10 5 cells/cm 2 in 24 well plates (Costar) or on wells pre-coated with Dormacell 1.2 microcarriers. At 0, 1, 2 or 3 days post-plating, the cells were separately infected with the recombinant penton base and fiber pBlueBac II vectors prepared above at a MOI of 5-30 pfu/cell. The cell concentration used to calculate MOI after 1, 2 and 3 days of cell growth was estimated by using the cell doubling time of 24 hours measured for Tn 5 cells in tissue culture flasks. In the other culturing conditions, Tn 5 cells were also infected with the recombinant vectors of this invention using an MOI of 5-30 pfu/cell.
  • T-25 flasks were seeded with Tn 5 cells at a density of 2 X 10 6 cells/culture.
  • the flasks were then infected with the high titer viral stock for both recombinant viruses at a MOI of 5.
  • Aliquots of cells were removed over a period of 5 days and analyzed for the presence of either penton base or fiber.
  • larger scale protein expression was then performed in T-150 flasks seeded with cells at a density of 2 X 10 6 cells/ml and infected with the viral stock to a MOI of 5.
  • the infected cells were then harvested by centrifugation at 4 days post-infection.
  • the supernatant was transferred to a fresh tube and stored at 4°C.
  • the pellet was stored at -20°C.
  • the cells were separately resuspended in 20 ml of 40 mM Tris-Hcl at pH 7.65 containing the protease inhibitors (aprotinin at 1 ⁇ g/ml, PMSF at 2 mM and leupeptin at 2 ⁇ g/ml) .
  • the suspension was then vortexed and placed on ice for 10 minutes to form a cell lysate.
  • the cell lysate was then centrifuged at 15,000 X g to pellet cell debris and form a recombinant protein-containing supernatant.
  • Fiber protein was purified from the resultant supernatant by DEAE Sepharose (Pharmacia, Piscataway, NJ) .
  • the running buffer for the column chromatography was 40 mM Tris-Hcl buffer at pH 7.65.
  • the supernatants were applied to the column and the recombinant fiber protein was then eluted from the column with a linear gradient of 10 mM to 500 mM NaCl.
  • Recombinant fiber protein eluted at 70 mM NaCl.
  • the peak fractions were collected and pooled.
  • the recombinant fiber protein was concentrated by Amicon filters.
  • the concentrated recombinant fiber protein was then further purified by chromatography over a Superose 6B column by FPLC (Pharmacia) according to manufacturer's instructions. Approximately 1-5 milligrams (mg) of recombinant fiber of greater than 90% purity was obtained following this purification procedure from 3 T-150 flasks. A single subunit of the purified individual protein was 62 kilodaltons (kD) (582 amino acids) under reducing SDS-PAGE. The fiber protein exists as a trimer of these three identical polypeptides with a resulting molecular weight of 180 kD.
  • Recombinant penton base was purified as described above for the fiber protein with the only exception that the penton base eluted from the DEAE Sepharose column at 170 mM NaCl following 2 washes with 80 mM NaCl.
  • a single subunit of the purified recombinant penton base was 82 kD under reducing SDS-PAGE.
  • Approximately 50 mg of recombinant penton base having a purity of greater than 90% was purified using the above procedure from 3 T-150 flasks.
  • Penton base exists as a pentamer consisting of 5 identical single subunits that are noncovalently associated to form a ring-shaped complex of approximately 90 A in diameter with a central cavity of approximately 10-20 A.
  • KB cells are maintained at 3 to 5 x 10 5 cells in Eagle's basal medium supplemented with 5% horse serum and infected with a MOI of 50 particles per cell.
  • Cells are harvested 32 hours post-infection and washed three times in phosphate-buffered saline. The resultant cell pellet is resuspended in ten volumes hypotonic 0.01 M Tris-HCl buffer at pH 8.1, 1 mM EDTA and subjected to five cycles of quick freezing and thawing.
  • the cell lysate is then mixed in a Waring-blendor with an equal volume of fluorocarbon Freon 113.
  • the aqueous phase obtained by low-speed centrifugation containing the viral material is centrifuged for 1 hour at 20,000 rev/min on a caesium chloride cushion (density, 1.43 g/ml) in a swinging bucket rotor (Beckman SW 27) .
  • Mature virions appear as a visible opalescent band at the top of the CsCl cushion.
  • the band containing the virions is collected and the virus particles are further purified by density-gradient centrifugation in caesium chloride.
  • the supernatant above the virion band in the two successive cycles of centrifugation is the source of adenovirus-soluble antigens, namely penton base and fiber proteins.
  • a saturated solution of ammonium sulfate at 4°C is added to the supernatant to a final degree of 55% saturation and the precipitate is allowed to form for 15 hours at 4°C and pH 6.5.
  • the precipitate formed is centrifuged at 5000 x g for 20 minutes at 4°C and the pellet is dissolved in the minimum volume of 0.05 M sodium phosphate buffer pH 6.8 and dialyzed for 72 hours in the cold against 10 volumes of the same buffer with at least five changes.
  • the viral antigen solution is cleared of possible incomplete low-density virus particles present in the centrifugation supernatant by centrifugation at 110,000 x g for 2 hours and the final supernatant or crude antigen preparation stored at -20°C until further fractionation.
  • a volume of crude preparation corresponding to 250 to 300 mg protein is loaded on top of the column and the gel is rinsed with 100 ml of the equilibrating buffer. This is then applied a linear sodium chloride gradient in the 0.05 M sodium phosphate buffer at pH 6.8, ranging from 0.0 to 0.5 M (500-ml total volume) .
  • the column is rinsed with 30 ml equilibrating buffer, then a 0.01 to 0.30 M (for penton and hexon) or 0.01 to 0.50 M (for fiber) linear gradient of potassium phosphate at pH 6.8 is applied in a total volume of
  • Native samples are electrophoresed at 5 mA per tube (in disc gel) or 2.5 mA per slot (in slab gel) in 6% polyacrylamide gel (acrylamide:bisacrylamide ratio, 30:0.8) buffered with 0.375 M Tris-HCl at pH 8.9 and overlayered with a spacer gel made of 3% polyacrylamide (acrylamide:bisacrylamide ratio, 10:2.5) in 0.125 M Tris-HCl buffer at pH 6.8.
  • the electrode buffer is 0.05 M Tris-0.384 M glycine at pH 8.3.
  • Polypeptide analysis is carried out in sodium dodecyl sulfate (SDS) -containing polyacrylamide gel.
  • Penton Complex with Purified or Recombinant Fiber and Penton Base
  • a recombinant penton complex consisting of recombinant penton base and fiber was prepared.
  • the purified recombinant penton base and fiber proteins prepared in Example 1 were combined in a 1:1 molar ratio in PBS at 4"C and maintained overnight.
  • the resultant recombinant penton complex consisting of 5 subunits of penton base and 3 fiber subunits per penton base were subsequently purified away from noncomplexed recombinant penton base or fiber proteins over linear sucrose gradients.
  • Penton complex is also prepared by combining Ad2-purified penton base and fiber proteins prepared in Example 2.
  • the Ad2-purified proteins of this invention can be used in gene delivery systems instead of the recombinant proteins with equivalent success.
  • either the recombinant or the Ad2-purified proteins of this invention can be used to promote successful gene delivery of exogenous genes.
  • the confirmation of the formation of a complex between recombinant penton base and fiber was performed using by immunoprecipitation using a monoclonal antibody directed against the fiber protein.
  • the procedure used was essentially as follows: First, purified adenovirus penton base was metabolically labeled with 35 S-methionine and purified on DEAE-Sepharose as described above. The labeled penton base was then added to unlabeled fiber to form a labeled complex that was immunoprecipitated by the anti-fiber monoclonal antibody FblO. The complex was analyzed on 10% SDS gel and autoradiographed.
  • Example 4 DNA Delivery Into Mammalian Cells DNA transfer has been accomplished by means of receptor-mediated endocytosis pathway as described by Wu et al., J. Biol. Chem. 262:4429-4432 (1987) and Wagner et al. , Proc. Natl. Acad. Sci. , USA 7:3410-3414 (1990) .
  • the use of this cellular-mediated gene transfer system has certain advantages in that it is not toxic to the recipient eucaryotic cell membrane, DNA can be transferred repetitively and the DNA can be targeted to specific cells by the distribution of cell-specific receptors.
  • bifunctional molecular conjugate gene transfer vehicles that are synthetically derived are used.
  • One such bifunctional conjugate is transferrin and poly-L-lysine where the transferrin ligand binds to transferrin receptors on the surface of recipient cells and is then internalized resulting in the cotransportation of the poly-L-lysine that is a DNA-binding moiety.
  • Adenovirus has been shown to enter cells in a similar manner as that of ligand-DNA complexes but they have the capacity to disrupt the endosome thereby escaping lysosomal destruction in the cytoplasm. See, Seth et al. , J. Virol. 51:650-655 (1984) and Seth et al. , Mol. Cell. Biol. 4:1528-1533 (1984) . More recently, replication-deficient adenovirus has been shown to augment the gene transfer of transferrin-polylysine conjugates mediated by the adenovirus-disruption of the endosome as described by Curiel et al. , Proc. Natl. Acad.
  • penton base and fiber proteins of this invention to mediate the transfer of foreign genes into recipient cells both in vi tro and in vivo overcomes the limitations of the above-described gene transfer systems.
  • This invention utilizes a recombinant coat protein subunit of adenovirus, the penton, which duplicates the cell receptor binding and DNA delivery properties of intact adenovirus virions and thus represents an improved method for gene therapy as well as for antisense-based antiviral therapy.
  • the ability of penton base and fiber to mediate the transfer of exogenous or non-native genes into cells was first evaluated in an in vi tro tissue culture system.
  • the DNA for transfer in the in vi tro assay was a DNA plasmid containing the luciferase gene, pRSVLuc.
  • the ligand-DNA complex, transferrin-poly-L-lysine, was also used in each transfer experiment as it allowed for the detection of gene transfer from the cell endosome to the cytoplasm. Luciferase activity was then assayed at 48 hours post transfection.
  • DMEM Dulbecco's modified Eagle's medium
  • penicillin at 100 international units/ml
  • streptomycin at 100 ⁇ g/ml and 2 mM glutamine.
  • the DNA plasmid, pRSVLuc containing the Photinus pyrali ⁇ luciferase gene under the control of the Rous sarcoma virus long terminal repeat enhancer/promoter was used as a reporter gene.
  • the pRSVLuc was prepared as described by DeWet et al., Mol. Cell. Biol.
  • reporter genes such as those expressing ⁇ -galactosidase, alkaline phosphatase and chloramphenicol acetyltransferase are contemplated for use in vi tro gene transfer experiments as described herein and are well known to one of ordinary skill in the art.
  • transferrin-poly-L-lysine-pRSVLuc complexes also referred to as conjugate-DNA complexes
  • 6 ⁇ g of pRSVLuc DNA in 350 ⁇ l of HBS 150 mM NaCl and 20 mM Hepes at pH 7.3
  • 12 ⁇ g of the transferrin-poly-L-lysine conjugate diluted in 150 ⁇ l of HBS complexes were allowed to form for 30 minutes at room temperature before admixing with cells.
  • the cells prepared as described above were grown in 10 centimeter tissue culture plates until approximately 50% confluent (5 X 10 6 cells) . Medium was removed and 1 ml of DMEM or Eagle's minimal essential medium containing 2% fetal bovine serum was then added. Conjugate-DNA complexes prepared above were then admixed with the cells followed by admixture of 50 ⁇ g of either recombinant penton base, recombinant fiber or both recombinant proteins together.
  • the transferrin-poly-L-lysine without the pRSVLuc DNA or the recombinant proteins alone was added to the cells while in the other control, the conjugate-DNA complex was added in the absence of the recombinant proteins.
  • the plates were returned to the incubator (5% C0 2 at 37°C) after which 3 ml of complete medium was added. After an additional 48 hours, the cells were harvested by lysis by freezing and thawing cycles for analysis of luciferase gene expression on a luminescence photometer according to manufacturer's instructions (Analytical Luminescence Laboratory, San Diego, CA) .
  • DNA delivery into cells via the adenovirus penton base is illustrated. Luciferase activity was measured and plotted against sample number, as shown. The sample numbers are listed on the X-axis while luciferase activity (in light units) is plotted on the Y-axis.
  • the samples identified by numbers 1-5 contained the following: (1) transferrin-poly-L-lysine (shown as transferrin/PL) , but no pRSVLuc; (2) pRSVLuc + transferrin/PL; (3) pRSVLuc + transferrin/PL + penton base + fiber; (4) pRSVLuc + transferrin/PL + penton base; (5) pRSVLuc + transferrin/PL + fiber.
  • a DNA plasmid containing the luciferase gene, pRSVLuc was added to monolayers of HeLa cells which had been previously plated onto type IV collagen. DNA delivery was carried out in the presence of transferrin/poly L-lysine to allow detection of gene transfer from the cell endosome to the cytoplasm. Luciferase activity was assayed at 48 hours post transfection. (Refer to Example 3 for details of the gene transfer experiment.) The samples containing recombinant penton base both demonstrated successful DNA delivery into the cells as measured by luciferase activity. Sample 4, which contained recombinant adenovirus fiber as well as penton base, displayed a somewhat greater introduction of DNA into the cells.
  • the advantages of using the recombinant proteins of this invention as described in Example 4 are applicable in this context of both in vi tro and in vivo utility.
  • administration is accomplished by first isolating a selected cell population from a patient such as lung epithelial cells, lymphocytes and the like followed by in vi tro gene transfer of the therapeutic compositions of this invention and the replacement of the cells into the patient.
  • In vivo therapy is preferred through the administration of the therapeutic compositions of this invention by aerosol means.
  • Also contemplated for in vivo applications are methods of administering therapeutic compositions of this invention by subcutaneous, intravenous, intraperitoneal, intramuscular, ocular means and the like.
  • penton-mediated gene delivery may be based on coating the penton base or penton complex with poly-L-lysine, which allows formation of a complex with DNA as well as binding to cell receptors.
  • the low pH in the endosome causes penton base-mediated disruption of the endo ⁇ omal membrane and relea ⁇ e of the DNA plasmid into the cytoplasm.
  • penton ba ⁇ e or penton complex in facilitating the transfer of exogenous genes into the lungs as described above, other means are contemplated to achieve the same results in both in vivo and in vi tro applications.
  • One gene delivery system utilizing the recombinant adenovirus penton complex result ⁇ in the transfer of genes through the use of an avidin-biotin complexation reaction and an antibody bridge to either recombinant fiber, penton base or penton complex.
  • the required reagents include the following: either purified or recombinant-produced fiber and penton base proteins used separately or non-covalently bound into penton complex; a nonfunctional blocking antibody, preferably a monoclonal antibody, that immunoreacts with either fiber or penton base proteins so that it does not inhibit the protein's functional activity; a biotinylated CIAP (calf intestinal alkaline phosphatase expression vector) into which the exogenous or foreign gene or therapeutic DNA sequence of interest has been inserted; and a chimeric protein consisting of Protein A linked to streptavidin (referred to as SA-PA) .
  • SA-PA Protein A linked to streptavidin
  • the delivery of thi ⁇ gene into the cell allow ⁇ can be mea ⁇ ured by quantitative colorimetric assay ⁇ much in the same fashion as ⁇ -galactosidase and the like. Biotinylation of one nucleotide on the plasmid allows its binding to the SA-PA chimeric protein in a defined way. Each SA-PA molecule can bind 4 biotin moieties linked to DNA.
  • An expres ⁇ ion vector capable of producing SA-PA in bacteria has been previously described by Sano et al. , Science 258:120-122 (1992) and Sano et al. , Bio/Technology 9.:1378 (1991) .
  • Antibodies used in this invention include monoclonal and monospecific polyclonal antibodies that immunoreact with domains on the fiber or penton base proteins such that the protein's biologic functions are not inhibited. For example, see, Boudin et al. , Virol. 92: 125-138 (1979). Alternative monoclonals to nonfunctional domains of c.-v integrins such as LM142 described by Cheresh et al., J. Biol. Chem. 262:1434-1437 (1987) are also contemplated for used in this invention.
  • the biotinylated DNA plasmid containing the therapeutic DNA sequence of interest binds to the streptavidin-Protein A chimeric protein.
  • the DNA-bound SA-PA complex then binds via the PA moiety to an antibody that immunoreact ⁇ with either the fiber or penton base (also referred to as ligands) .
  • the ligands are either used separately in the noncomplexed form or used in the complexed form where fiber protein is noncovalently bound to penton base. If the fiber protein is used in the noncomplexed form to bind to an anti-fiber antibody, following the formation of the antibody and fiber bridge, the products are then admixed with penton base to form penton complex.
  • a penton complex (also referred to as penton) is ultimately formed irrespective of whether the proteins of this invention are used in the noncomplexed form initially.
  • the formed PA-immobilized antibody-ligand complex then binds, via the functional receptor-binding site on the ligand, with the ligand-specific receptor ⁇ expre ⁇ ed on the recipient cell ⁇ urface.
  • the resultant ligand-occupied receptors then provide for the delivery of the exogenous or foreign genes/DNA ⁇ equence(s) into the cell via the specific functions of the adenovirus-derived proteins of this invention.
  • the fiber protein binds to fiber-specific receptors the binding of which provides for nuclear localization signal targeting following receptor internalization.
  • the penton base protein that is either admixed separately with the fiber-antibody bridge-PA-SA-DNA complex or is already noncovalently bound to fiber prior to the formation of the antibody bridge complex, provides for the enhanced uptake of DNA into the cytoplasm via the release from the endosome.
  • penton base without fiber protein in the formation of the antibody-PA-SA-biotinylated DNA plasmid complex.
  • the amino acid residue sequence present in the penton base arginine-glycine-aspartic acid (RGD) is used to target the penton base to RGD-specific receptors expressed on cell surfaces.
  • RGD peptide sequence present in penton base has been shown to be the ligand binding site recognized by integrin receptors such as the vitronectin and fibronectin integrin receptors.
  • the recombinant fiber or penton base protein provides for targeting of the foreign gene to epithelial, endothelial, platelets and lymphoid cells expressing fiber-specific receptors.
  • the recombinant penton base enhances the uptake of foreign DNA and the release from the endosome evading destruction by lysosomal enzymes. If the gene transfers are performed on cultured cells in vi tro for the ultimate replacement into a patient requiring gene therapy, the cells can be plated on a synthetic RGD-coated surface that result ⁇ in the upregulation of the fibronectin receptor ⁇ (alpha 5 betaj) thereby augmenting adenovirus-protein mediated uptake.
  • targeting to nonepithelial cells is accomplished by plating those cells on RGD in the presence of high concentrations of manganese which upregulates fibronectin receptors that will bind penton base RGD.
  • RGD fibronectin receptors
  • Also contemplated for use in this invention are the alternative means of gene delivery where genes, DNA oligonucleotide sequences such as those encoding ribozymes, or antisense-based plasmids are conjugated directly to a region of the penton (penton base plus fiber) such that the gene delivery functions of the proteins are not inhibited.
  • the plasmid-based vectors can either be under the control of strong constitutive promoters or regulatable promoters along with enhancer element ⁇ to obtain higher levels of gene expression than that obtained by incorporating the therapeutic gene into the intact or replication-deficient genome.
  • Constitutive promoters include human cytomegalovirus which contains an enhancer sequence while regulatable promoters include those that are regulated by chemical ⁇ or temperature.
  • Chemically regulatable promoters for eucaryotic expres ⁇ ion systems include those based on induction by ⁇ -interferon, heat-shock, heavy metal ions, and steroids such as glucocorticoids as described by Kaufman et al. , In "Current Protocols in Molecular Biology” , Ausubel et al. , eds.
  • receptor-binding ⁇ ite pre ⁇ ent in ligands specific for other receptors onto the fiber protein by recombinant DNA techniques for the production of a chimeric protein molecule as described herein.
  • the chimeric molecule is referred to as a penton complex-ligand conjugate or a penton base-ligand conjugate depending on whether the protein ⁇ are u ⁇ ed in the complexed or noncomplexed form.
  • the chimeric molecule i ⁇ then complexed via the fiber protein to the anti-fiber antibody- (PA-SA) -biotinylated DNA containing the exogenou ⁇ DNA prepared as described above.
  • PA-SA anti-fiber antibody-
  • receptor ⁇ and their re ⁇ pective ligand ⁇ or attachment ⁇ equences for use in gene delivery include the following: CR2 receptor binding to the amino acid residue attachment sequences EDPGFFNVE (SEQ ID NO 6) and EDPGKQLYNVE (SEQ ID NO 7) ; CD4 receptor recognizing the V3 loop of HIV gpl20; transferrin receptor and its ligand transferrin; low density lipoprotein receptor (LDL) and its ligand; and asialoglycoproteins that recognize deglycosylated protein ligands.
  • LDL low density lipoprotein receptor
  • asialoglycoproteins that recognize deglycosylated protein ligands.
  • antibodies specific to preselected cell surface receptors can also be used in the gene delivery system described above.
  • Antibodies defined for use in this manner include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules such as those containing an antibody combining site or paratope.
  • Exemplary antibodies are intact immunoglobulin molecule ⁇ , substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab')2 and F(v) .
  • Antibodies can be produced by conventional techniques for preparing polyclonal and monoclonal antibodies and also by recombinant DNA techniques familiar to one of ordinary skill in the art and as described in US Patent 4,816,567 and International Application WO 90/14424.
  • Antibodies specific for cell surface receptors for use a ⁇ de ⁇ cribed herein include antibodie ⁇ to integrin ⁇ , MHC class I and clas ⁇ II, asialoglycoprotein receptor, transferrin receptor, LDL receptor and the like.
  • the gene delivery sy ⁇ tems utilizing the adenovirus-derived penton base and fiber proteins of this invention are exemplary means for therapeutic intervention of common hereditary disorders involving respiratory epithelium including emphysema and cystic fibrosis.
  • this invention i ⁇ u ⁇ eful for the delivery of anti ⁇ en ⁇ e compound ⁇ to epithelial cell ⁇ . Since a large number of human pathogen ⁇ including rhinoviru ⁇ e ⁇ , rotoviruses, herpesviruses, and papilloma virus invade the ho ⁇ t via the epithelial cell route as a primary site of infection, the ability to target antiviral agents to epithelial cells is of therapeutic importance.
  • the use of adenovirus-derived penton gene delivery system ⁇ i ⁇ also valuable for the delivery of immunotoxin for the treatment of various cancers, including melanomas or osteosarcomas.
  • the present invention contemplates that the therapeutic compounds and compositions of the present invention may be directed to specific receptors or cells, for the ultimate purpose of delivering those compounds and compositions to specific cells or cell type ⁇ .
  • the adenovirus-derived proteins and polypeptides of the present invention are particularly useful in this regard.
  • Adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and ⁇ v integrin receptors for internalization.
  • adenovirus attachment to the cell surface via the fiber coat proteins has now been discovered to be dissociable and distinct from the subsequent step of internalization, an event that depends on ⁇ v integrin binding to the RGD-containing penton base viral coat protein.
  • Integrins which recognize the RGD motif include ⁇ 5 ⁇ 1; c. Ilb ⁇ 3 , and most, if not all, ⁇ v -containing integrin ⁇ (i.e., vitronectin receptors). Integrins have also been implicated in the attachment of certain viruses and bacteria to host cells. For example, the collagen/laminin-binding integrin, ot 2 ⁇ , has been identified as a receptor for echovirus 1 attachment
  • Vitronectin receptor is an integrin that mediates cell adhesion and other cellular receptor functions. Vitronectin receptor i ⁇ a heterodimeric receptor defined by the pre ⁇ ence of an ⁇ v ⁇ ubunit and a second, ⁇ subunit, typically / ⁇ 3 or ⁇ s .
  • Ligands for the vitronectin receptor are arg-gly-asp (RGD) -containing polypeptide ⁇ or protein ⁇ , ⁇ uch as the adenovirus penton base.
  • integrins may be classified in a relatively simple fa ⁇ hion ba ⁇ ed on their binding characteri ⁇ tic ⁇ , as shown in Table 1.
  • Integrins that bind primarily to basement membrane proteins 1 Integrins that bind primarily to matrix proteins of inflammation, wound healing, and development
  • the binding interaction between penton base and the vitronectin receptor is RGD-dependent .
  • an adenovirus penton ba ⁇ e protein, and polypeptide ⁇ derived therefrom which contain at least the RGD domain of the penton base protein are expected to be useful in directing the therapeutic nucleotide sequences of the present invention to the vitronectin receptor.
  • the invention also contemplates isolated penton base protein, penton base fragments, and penton base-derived polypeptides which contain the RGD domain of adenovirus penton base.
  • isolated penton base protein de ⁇ cribed herein produced by recombinant DNA methods or produced by purification from adenovirus ⁇ tock sources, (2) polypeptides having the RGD sequence and that substantially bind vitronectin receptor, and (3) compositions comprising one or more of the above ligands.
  • the pre ⁇ ent invention contemplate ⁇ the u ⁇ e of any vitronectin receptor ligand a ⁇ an active ingredient in a therapeutic compo ⁇ ition according to the present invention, although adenovirus penton-derived proteins or polypeptides are particularly preferred.
  • a vitronectin receptor ligand can be any polypeptide, protein, polypeptide or protein derivative, fragment or analog thereof which has the ability to substantially bind to vitronectin receptor.
  • Substantially bind in the present context means a binding affinity sufficient to be useful in the methods of the present invention.
  • useful competition for binding to a receptor depends on both the binding affinity and the concentration of ligand achievable in the vicinity of the receptor.
  • binding affinities lower than that found on a natural ligand such as isolated adenovirus penton base are useful, so long as the cell or tissue to be treated can tolerate concentrations of ligand sufficient to compete with adenovirus binding to the receptor.
  • Natural binding affinity of isolated penton ba ⁇ e was determined to have a dis ⁇ ociation con ⁇ tant (Kd) of 55 nanomolar.
  • a preferred adenoviru ⁇ -derived protein or polypeptide has a sequence that correspond ⁇ to adenovirus strain 2 or 5 (Ad2 or Ad5) .
  • Another preferred adenovirus-derived protein or polypeptide for use as a vitronectin receptor ligand according to the present invention includes the sequence represented by the formula -HAIRGDTFA- (SEQ ID NO 1) .
  • Synthesis of useful polypeptides according to the present invention can be accomplished by a variety of well known methods, including the procedures described hereinabove.
  • Method ⁇ for identifying other suitable vitronectin receptor ligands u ⁇ eful in the present compositions such as additional polypeptides based on the sequence of an adenovirus' penton base amino acid residue sequence, can be identified by the use of the cell binding ⁇ ssays described in the above Examples.
  • the penton base coat protein contains five RGD sequence ⁇ and wa ⁇ thus postulated to interact with cell surface integrins.
  • cell adhesion experiments were performed on surfaces coated with various cell matrix proteins or with recombinant penton ba ⁇ e.
  • the cell adhe ⁇ ion a ⁇ ay to extracellular matrix protein ⁇ and to recombinant Ad2 penton base was performed using three different cell lines.
  • the M21 melanoma cell line was obtained from Dr. Donald Mortan (University of California at Los Angeles, CA) .
  • M21-L4 ( ⁇ v -expressing) and M21-L12 (c_ v -deficient) cell lines were derived from the M21-L cell line as described by Cheresh et al . , J. Biol. Chem. 262: 17703-17711 (1987) and characterized for their integrin expression as previously described by Felding-Habermann et al . , J. Clin. Invest. 89: 1-5
  • HeLa cells ATCC Accession No CCL2
  • A549 lung carcinoma cell line ATCC Acce ⁇ ion No CCL185
  • ATCC American Type Culture Collection
  • the cells were first labeled with 3 H-thymidine.
  • Individual wells of 48 well non-treated cluster plates at a concentration of 5 X 10 4 cells/well (Costar, Cambridge, MA) were first pre-coated overnight with varying amounts of extracellular matrix proteins or penton base.
  • Vitronectin, laminin, fibronectin, and collagen I were purcha ⁇ ed from Sigma (St. Loui ⁇ , MO).
  • Recombinant penton base was prepared as described in Example 1.
  • the wells were separately coated with vitronectin (10 ⁇ g/ml) , collagen I (10 ⁇ g/ml) , laminin (10 ⁇ g/ml) , or penton base (0.5 ⁇ g/ml) .
  • the coated wells were then blocked with 5% BSA in PBS at pH 7.4 for 1 hour.
  • the versene-relea ⁇ ed labeled HeLa, ,M21, or A549 cell ⁇ re ⁇ uspended in adhesion buffer (DMEM supplemented with 2 mM MgCl 2 , 1% BSA, and 20 mM HEPES) were then added and allowed to attach at 37°C for 1 hour, the time at which adhesion reached a maximum in wells containing the most penton base and vitronectin.
  • Unattached cells were removed by rapid washing with PBS and the amount of cell-a ⁇ ociated radioactivity remaining in each well was determined by addition of detergent and scintillation counting the cell lysate ⁇ . The percentage of attached cell ⁇ wa ⁇ calculated from the total cell cpm added to each well.
  • HeLa, M21, and A549 cell ⁇ efficiently attached to vitronectin, collagen and laminin.
  • these cells also attached to and spread on recombinant penton base-coated wells.
  • the wells were coated with 0.5 ⁇ g/ml penton base or 10 ⁇ g/ml of vitronectin, laminin, or collagen I and then blocked with BSA.
  • Cells were pretreated with 150 ⁇ g/ml of synthetic peptides (RGD- containing or non-RGD-containing, e.g., GRGDSP or GRGESP) in adhesion buffer for 1 hour at 4°C and then allowed to attach to the wells at 37°C for 30-60 minute ⁇ .
  • the percentage of attached cells wa ⁇ determined a ⁇ above.
  • Synthetic peptide ⁇ useful as described above are commercially available.
  • RGD-containing (e.g., GRGDSP) and non-RGD-containing (e.g., GRGESP) peptides are available from Telio ⁇ Pharmaceutical ⁇ , Inc. (San Diego, CA) .
  • synthetic peptides may be prepared via well-known techniques, such as the classical solid-phase technique described by Merrifield, Adv. Enzvmol. 32: 221-296 (1969) as adapted for use with a model 430 automated peptide synthesizer (Applied Biosystems, Foster City, CA) .
  • M21-L cell ⁇ ⁇ pecifically lack ⁇ v mRNA and protein a ⁇ de ⁇ cribed by Chere ⁇ h et al. , J. Biol. Chem. 262: 17703-17711 (1987) .
  • the cell ⁇ were prepared as described in Section A immediately above and tran ⁇ fected with a full length ⁇ v cDNA, de ⁇ ignated M21-L4 or mock-transfected, designated M21-L12.
  • M21-L4 cells expressing ⁇ v attach to vitronectin using ⁇ v j ⁇ 3 and ⁇ v/ 3 5 while M21-L12 cell ⁇ , which fail to express these integrins, do not (Felding-Habermann et al. , J___ Clin. Invest. 89: 1-5 (1992) .
  • Both cell line ⁇ expre ⁇ ⁇ _ integrin ⁇ and thus attach to collagen, laminin, and fibronectin (Felding-Habermann et al. , ⁇ upra .
  • M21-L4 or M21-L12 Adhesion of M21-L4 or M21-L12 to polystyrene wells precoated with varying doses of either vitronectin or penton base was examined.
  • M21-L4 ( ⁇ v +) but not M21-L12 ( ⁇ v -) cells adhered to vitronectin in a dose-dependent manner while both cell types bound to collagen I (data not shown) .
  • ⁇ v integrins were used to examine cell interaction with penton base or adenovirus infection of cells.
  • M21-L4 ( ⁇ v +) cells were incubated with function-blocking MAbs to the vitronectin receptors, ⁇ v /3 3 (LM609) and ⁇ v /3 5 (P3G2) , a control, non-function-blocking MAb to the c_ v subunit of ⁇ v integrins (LM142) or with an RGD-containing peptide and then examined for adhesion to penton base-coated surfaces.
  • MAbs monoclonal antibodies
  • the LM142 monoclonal antibody (MAb) directed against a nonfunctional epitope of v integrins was produced as previously described (Cheresh et al . , J. Biol. Chem. 262: 1434-1437 (1987) .
  • HE-p-2, HeLa and H2921 cell lines were obtained from ATCC (Rockville, MD) .
  • the CS-1 hamster melanoma cell line (Farishian, et al . , Arch. Biochem. Biophvs . 198 : 449-461 (1979)) , a generous gift of Dr. Carolyn Da ⁇ ky (UCSF, San Francisco, CA) wa ⁇ propagated in RPMI supplemented with 10% fetal bovine serum, 2mM L- glutamine, and 50 ⁇ g/ml gentamicin.
  • CS-1 cells do not adhere to vitronectin due to their failure to expres ⁇ ⁇ v integrins (Thomas, et al. , J. Cell. Sci. 105: 191- 201 (1993)). These cells express neither ⁇ nor ⁇ v ⁇ 5 heterodimers on their ⁇ urface yet maintain an intracellular pool of the ⁇ v integrin ⁇ ubunit.
  • cDNAs encoding full-length ⁇ 3 (Fitzgerald, et al., J. Biol. Chem.
  • subunit proteins were ⁇ ubcloned into the pcDNA-1/NEO expression vector (Invitrogen, La Jolla, CA) and introduced into CS-1 cells via lipofectin- mediated transfection (Gibco-BRL, Gaithersburg, MD) .
  • Stably transformed cells were selected by growth in 500 ⁇ g/ml Geneticin (Sigma, St.
  • Ad2 Human adenovirus type 2 was propagated in HeLa or H2981 cells and was purified and stored as previously described (Everitt, et al. , J. Virol. 21: 199-214 (1977)). Briefly, Ad2 wa ⁇ banded on ce ⁇ ium chloride gradients and then dialyzed against lOmM phosphate-buffered saline (PBS), pH 8.0, containing 10% glycerol and ImM MgCl 2 . Purified virus was ⁇ tored at -70°C and dialyzed into the appropriate buffer just prior to use. Ad2 was labeled with [ 3 H] -thymidine as previously described (Svens ⁇ on, et al. , J. Virol. 38: 70-81 (1981)) or with NHS-SS-biotin (Pierce, *) a ⁇ recommended by the manufacturer.
  • LM142 monoclonal antibody directed again ⁇ t a nonfunctional epitope of ⁇ v integrin ⁇
  • function-blocking mAbs LM609, P3G2, and P4C10 directed to ⁇ , ⁇ v ⁇ 5 , and w respectively, were produced as previously described (Cheresh, et al., J. Biol. Chem. 262: 17703-17711 (1987) ; Wayner, et al., J. Cell. Biol. 113: 919-929 (1991)) .
  • Binding of 3 H-thymidine-labeled Ad2 or 35 S-penton base to CS-l: ⁇ 3 and CS-l: ⁇ 5 cells was carried out as previously de ⁇ cribed (Wickham, et al . , Cell 73 : 303- 313 (1993)) . Nonspecific binding was determined in the presence of 50-fold exces ⁇ unlabeled Ad2 or penton base.
  • Ad2 internalization into CS-l: ⁇ 3 and CS-l: ⁇ 5 cells was performed with biotinylated Ad2 using a capture ELISA as previously described by Smythe, et al. (Meth. Enzvmol . 219: 223-234 (1992)) . Briefly, 60 ⁇ g of biotinylated Ad2 was added to 1 X 10 7 CS-l:f ⁇ or CS-l-. ⁇ 5 cells for 1 hour at 4°C in adhe ⁇ ion buffer. The unbound viru ⁇ wa ⁇ removed by wa ⁇ hing, and cell ⁇ amples of 1 X 10 6 cells each were warmed to 37°C for varying length ⁇ of time.
  • Uninternalized viru ⁇ particle ⁇ remaining on the cell surface were then "quenched" by the addition of lOO ⁇ g/ml of soluble avidin (Boehringer-Mannheim, Indianapoli ⁇ , IN) for 60 minute ⁇ at 4°C in HEPES-buffered saline containing lOmM EDTA. Internalized Ad2 was then released by solubilizing the cells with 1% NP-40 in PBS/0.2% of BSA. Cell lysate ⁇ containing biotin-Ad2 were then added to ELISA wells which had been precoated with l ⁇ g/well of polyclonal anti-penton base IgG.
  • Biotin- Ad2 was then detected by addition of ⁇ treptavidin- alkaline phosphatase diluted 1:1000 in PBS/0.5% nonfat dry milk (Blotto) followed by chromogenic sub ⁇ trate.
  • the total amount of Ad2-biotin bound to the cell ⁇ wa ⁇ determined at 4°C in the ab ⁇ ence of ⁇ oluble avidin.
  • Non ⁇ pecific binding was determined by analyzing cells incubated in the absence of biotin-Ad2.
  • radiolabeled penton base was added to cells at 4°C and then immediately warmed to 37°C.
  • samples were taken and diluted 10-fold in ice-cold HBSE (Hepes-buffered saline containing lOmM EDTA) , washed twice in thi ⁇ buffer, and then re ⁇ uspended in a small volume of HBSE containing 2mg/ml subtili ⁇ in (Sigma, St. Loui ⁇ , MO) and incubated at 37°C for 15 minutes. Finally, the cells were washed in ice-cold adhesion buffer, and the remaining protease-resi ⁇ tant Ad2-a ⁇ ociated cpm were mea ⁇ ured.
  • HBSE Hepes-buffered saline containing lOmM EDTA
  • samples were taken and diluted 10-fold in ice-cold HBSE (Hepe ⁇ -buffered saline containing lOmM EDTA) , washed twice in this buffer, and then resuspended in a small volume of HBSE containing 2mg/ml subtilisin (Sigma) and incubated at 37°C for 15 minutes, finally, the cells were washed in ice-cold adhesion buffer, and the remaining protease-resistant Ad2- associated cpm were measured. To determine the total cell as ⁇ ociated penton base, the samples were washed three times in HBS containing ImM CaCl 2 and ImM MgCl 2 . Subtilisin-treated cells remained >95% viable.
  • HBSE Hepe ⁇ -buffered saline containing lOmM EDTA
  • the cell samples were washed once with saline and then incubated for 1 hour at 37°C with 200ml permeability buffer 50mM MES (Sigma) -buffered saline, pH 6.0, containing 0.2% BSA, ImM CaCl 2 , ImM MgCl 2 , and 50mM NaN 3 .
  • 50mM MES Sigma
  • pH 6.0 a combination of 25mM MES and
  • 25mM HEPES was used to adjust the permeability buffer to the desired pH. After incubation in permeability buffer, the cell sample ⁇ were incubated with or without adenoviru ⁇ and then gently centrifuged. The percent 3 H-choline release was determined by mea ⁇ uring the count ⁇ released into the permeability buffer and the counts remaining in the cell pellet.
  • 3 H- choline release was determined by measuring the radioactivity in the buffer and in the adherent cells which were solubilized in 0.2ml of 1% SDS.
  • antibodie ⁇ or other soluble proteins were incubated with adherent cells for 1 hour prior to virus binding and in the subsequent virus binding and low pH incubations.
  • b. Result ⁇ In initial ⁇ tudie ⁇ , the potential involvement of o; v integrin ⁇ in Ad2-induced membrane permeabilization wa ⁇ analyzed.
  • CS-1 cells expressing ⁇ (CS-l: ⁇ 3) or ⁇ v ⁇ 5 (CS01: ⁇ 5) were established by transfecting CS-1 cells with cDNAs encoding the ⁇ 3 or ⁇ 5 subunit. Transfected cells were capable of adhering to immobilized vitronectin or penton base (not ⁇ hown) . Adhe ⁇ ion to penton ba ⁇ e or vitronectin wa ⁇ blocked by the appropriate mAb to ⁇ v ⁇ 3 (LM609) or ⁇ v ⁇ 5 (P3G2) but not by a control antibody to ⁇ l integrins (P4C10) . The parental cell line, CS-1, failed to adhere to either penton ba ⁇ e or vitronectin (data not shown) . (See also Thoma ⁇ , et al. , J. Cell. Sci. 105: 191-201 (1993) .)
  • M21-L4 cells express a 20- fold higher level of ⁇ than ⁇ v ⁇ 5 , these re ⁇ ult ⁇ suggested that penton base binding at low pH was mediated by ⁇ . v ⁇ 5 .
  • the binding of penton base to M21-L4 at low pH was not inhibited by RGD peptides, which suggests that the RGD sequence may have a limited role in binding at low pH.
  • therapeutic agents e.g., therapeutic nucleotide sequence ⁇
  • therapeutic agents may be designed to specifically target particular cell or tis ⁇ ue type ⁇ via the alteration and refinement of particular parameter ⁇ . For instance, variation of pH conditions in the delivery vehicle -- particularly if said vehicle is a lipo ⁇ ome -- may preferentially direct the vehicle to cell ⁇ expre ⁇ ing a particular receptor and may further facilitate entry of the vehicle and it ⁇ attached or enclo ⁇ ed therapeutic agent into the cell.
  • admini ⁇ tration of a therapeutic agent in conjunction with a delivery vehicle in a low pH environment may promote the interaction of the agent and vehicle (e.g., penton ba ⁇ e + therapeutic nucleotide sequence + liposomes) with cells expressing vitronectin receptors, particularly c. v ⁇ 5 receptors.
  • agent and vehicle e.g., penton ba ⁇ e + therapeutic nucleotide sequence + liposomes
  • vitronectin receptors particularly c. v ⁇ 5 receptors.
  • vitronectin receptor ligands such as the penton base are expected to be particularly useful in delivering therapeutic agents to, and into, integrin-expres ⁇ ing cell ⁇ -- particularly, cell ⁇ expressing vitronectin receptors. Further, in view of the finding that different integrin ⁇ , including vitronectin receptor ⁇ , are expressed on different cell types and even in different locations on cells, this information i ⁇ useful in designing targeting and delivery agent ⁇ po ⁇ e ⁇ sed of an elegant selectivity.
  • compositions including penton base and a therapeutic agent may be preferentially targeted to integrin receptor-expressing cells, such as matrix proteins involved in inflammation, wound healing, and development. More particularly, such a composition is contemplated to be useful in targeting cells expressing ⁇ or ⁇ v ⁇ 5 integrins, or both.
  • composition ⁇ is useful in the treatment of inflammatory disorder ⁇ such as lupus erythematosu ⁇ , rheumatoid arthriti ⁇ , and the like, ⁇ uch compo ⁇ ition ⁇ would be u ⁇ eful in targeting ⁇ pecific malignant, tumorigenic and tran ⁇ formed cells.
  • integrin ⁇ have been implicated in tumor progre ⁇ ion and meta ⁇ ta ⁇ i ⁇ (Damjanovich, et al., Am J. Re ⁇ pir. Cell Mol. Biol. 6: 197-206 (1992) ; Ruo ⁇ lahti, et al .
  • integrins e.g. laminin, collagen, fibronectin
  • e.g. laminin, collagen, fibronectin are thus contemplated to be useful as disclosed herein, as well.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

La présente invention se rapporte à la thérapie génique, et, en particulier, à des procédés et des agents thérapeutiques permettant de cibler et de transporter des séquences nucléotidiques non natives vers des cellules spécifiques. Selon ces procédés, un penton dérivé d'un adénovirus, un complexe de penton, ou un penton-fibre sont utilisés pour faciliter le ciblage et le transport.
PCT/US1994/001263 1993-02-09 1994-02-03 Ciblage et transport de genes et d'agents antiviraux dans des cellules par l'adenovirus penton Ceased WO1994017832A1 (fr)

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AU61333/94A AU6133394A (en) 1993-02-09 1994-02-03 Targeting and delivery of genes and antiviral agents into cells by the adenovirus penton

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US1522593A 1993-02-09 1993-02-09
US08/015,225 1993-02-09
US4615993A 1993-04-13 1993-04-13
US08/046,159 1993-04-13

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WO1995026412A1 (fr) * 1994-03-28 1995-10-05 The Uab Research Foundation Ligands ajoutes a la fibre d'adenovirus
WO1996007734A3 (fr) * 1994-09-08 1996-05-09 Genvec Inc Adenovirus recombine comprenant une proteine chimerique a base pentonique
WO1996026281A1 (fr) * 1995-02-21 1996-08-29 Genvec, Inc. Proteine fibreuse chimere d'adenovirus et ses procedes d'utilisation
WO1996021007A3 (fr) * 1994-12-30 1996-09-06 Viagene Inc Systemes de transfert genique a mediation par bacteriophage capables d'effectuer une transfection des cellules eucaryotiques
WO1996029423A1 (fr) * 1995-03-20 1996-09-26 Baylor College Of Medicine Compositions et methodes pour induire une infection par des vecteurs retroviraux en dehors de leur gamme d'hotes
EP0696206A4 (fr) * 1993-08-13 1997-03-05 Genetic Therapy Inc Adenovirus contenant des proteines fibreuses modifiees
FR2741087A1 (fr) * 1995-11-13 1997-05-16 Commissariat Energie Atomique Complexe proteique dodecaedrique adenoviral, procede de preparation, composition le contenant et ses applications
WO1997018317A1 (fr) * 1995-11-13 1997-05-22 Commissariat A L'energie Atomique Complexe proteique dodecaedrique adenoviral, composition le contenant et ses applications
US5652224A (en) * 1995-02-24 1997-07-29 The Trustees Of The University Of Pennsylvania Methods and compositions for gene therapy for the treatment of defects in lipoprotein metabolism
FR2747681A1 (fr) * 1996-04-18 1997-10-24 Commissariat Energie Atomique Complexe proteique dodecaedrique adenoviral, composition le contenant et ses applications
US5756283A (en) * 1995-06-05 1998-05-26 The Trustees Of The University Of Pennsylvania Method for improved production of recombinant adeno-associated viruses for gene therapy
WO1998044121A1 (fr) * 1997-04-02 1998-10-08 Transgene S.A. Fibre adenovirale modifiee et adenovirus cibles
FR2761688A1 (fr) * 1997-04-02 1998-10-09 Transgene Sa Fibre adenovirale modifiee et adenovirus cibles
US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US5856152A (en) * 1994-10-28 1999-01-05 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV vector and methods of use therefor
US5872154A (en) * 1995-02-24 1999-02-16 The Trustees Of The University Of Pennsylvania Method of reducing an immune response to a recombinant adenovirus
WO1999023237A1 (fr) * 1997-11-03 1999-05-14 Commissariat A L'energie Atomique Vecteur peptidique de transfection, composition le contenant et leurs applications
US5965541A (en) * 1995-11-28 1999-10-12 Genvec, Inc. Vectors and methods for gene transfer to cells
US6001557A (en) * 1994-10-28 1999-12-14 The Trustees Of The University Of Pennsylvania Adenovirus and methods of use thereof
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US6251677B1 (en) 1997-08-25 2001-06-26 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
US6251957B1 (en) 1995-02-24 2001-06-26 Trustees Of The University Of Pennsylvania Method of reducing an immune response to a recombinant virus
US6261551B1 (en) 1995-06-05 2001-07-17 The Trustees Of The University Of Pennsylvania Recombinant adenovirus and adeno-associated virus, cell lines, and methods of production and use thereof
US6274322B1 (en) 1991-09-30 2001-08-14 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
WO2002024934A1 (fr) * 2000-09-22 2002-03-28 Consorzio Interuniversitario Per Le Biotecnologie Vecteurs chimeres et leur utilisation pour le transfert de genes heterologues
US6372208B1 (en) 1999-09-28 2002-04-16 The Trustees Of The University Of Pennsylvania Method of reducing an immune response to a recombinant virus
US6387368B1 (en) 1999-02-08 2002-05-14 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
US6455314B1 (en) 1998-09-11 2002-09-24 Genvec, Inc. Alternatively targeted adenovirus
US6599737B1 (en) 1998-04-30 2003-07-29 Cornell Research Foundation, Inc. Adenoviral vectors with tandem fiber proteins
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US6974695B2 (en) 2000-11-15 2005-12-13 Crucell Holland B.V. Complementing cell lines
US7052881B2 (en) 1995-06-15 2006-05-30 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US7232899B2 (en) 1996-09-25 2007-06-19 The Scripps Research Institute Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
US7335509B2 (en) 1995-01-23 2008-02-26 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US7468181B2 (en) 2002-04-25 2008-12-23 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
US7611868B2 (en) 2003-05-14 2009-11-03 Instituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Recombinant modified adenovirus fiber protein
US7749493B2 (en) 1998-07-08 2010-07-06 Crucell Holland B.V. Chimeric adenoviruses
WO2011014794A1 (fr) 2009-07-31 2011-02-03 Paxvax, Inc. Vecteurs à base adénovirale
US7993672B2 (en) 1995-01-23 2011-08-09 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
WO2017039311A1 (fr) * 2015-08-31 2017-03-09 한양대학교 산학협력단 Composition pour l'administration intracellulaire contenant un peptide dérivé de la protéine vi des adénovirus et composition pharmaceutique contre le cancer contenant ce dernier

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US6274322B1 (en) 1991-09-30 2001-08-14 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
EP0696206A4 (fr) * 1993-08-13 1997-03-05 Genetic Therapy Inc Adenovirus contenant des proteines fibreuses modifiees
WO1995026412A1 (fr) * 1994-03-28 1995-10-05 The Uab Research Foundation Ligands ajoutes a la fibre d'adenovirus
WO1996007734A3 (fr) * 1994-09-08 1996-05-09 Genvec Inc Adenovirus recombine comprenant une proteine chimerique a base pentonique
US5731190A (en) * 1994-09-08 1998-03-24 Genvec, Inc. Penton base protein and methods of using same
US5712136A (en) * 1994-09-08 1998-01-27 Genvec, Inc. Adenoviral-mediated cell targeting commanded by the adenovirus penton base protein
US5559099A (en) * 1994-09-08 1996-09-24 Genvec, Inc. Penton base protein and methods of using same
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US5856152A (en) * 1994-10-28 1999-01-05 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV vector and methods of use therefor
US6203975B1 (en) 1994-10-28 2001-03-20 The Trustees Of The University Of Pennsylvania Adenovirus and method of use thereof
US5871982A (en) * 1994-10-28 1999-02-16 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
US6001557A (en) * 1994-10-28 1999-12-14 The Trustees Of The University Of Pennsylvania Adenovirus and methods of use thereof
WO1996021007A3 (fr) * 1994-12-30 1996-09-06 Viagene Inc Systemes de transfert genique a mediation par bacteriophage capables d'effectuer une transfection des cellules eucaryotiques
US5736388A (en) * 1994-12-30 1998-04-07 Chada; Sunil Bacteriophage-mediated gene transfer systems capable of transfecting eukaryotic cells
US7361640B2 (en) 1995-01-23 2008-04-22 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US7993672B2 (en) 1995-01-23 2011-08-09 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US7335509B2 (en) 1995-01-23 2008-02-26 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US6153435A (en) * 1995-02-21 2000-11-28 Cornell Research Foundation, Inc. Nucleic acid that encodes a chimeric adenoviral coat protein
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
WO1996026281A1 (fr) * 1995-02-21 1996-08-29 Genvec, Inc. Proteine fibreuse chimere d'adenovirus et ses procedes d'utilisation
US6576456B2 (en) 1995-02-21 2003-06-10 Cornell Research Foundation, Inc. Chimeric adenovirus fiber protein
US6887463B2 (en) 1995-02-24 2005-05-03 The Trustees Of The University Of Pennsylvania Methods and compositions for the treatment of defects in lipoprotein metabolism
US5872154A (en) * 1995-02-24 1999-02-16 The Trustees Of The University Of Pennsylvania Method of reducing an immune response to a recombinant adenovirus
US5652224A (en) * 1995-02-24 1997-07-29 The Trustees Of The University Of Pennsylvania Methods and compositions for gene therapy for the treatment of defects in lipoprotein metabolism
US6251957B1 (en) 1995-02-24 2001-06-26 Trustees Of The University Of Pennsylvania Method of reducing an immune response to a recombinant virus
US7306794B2 (en) 1995-02-24 2007-12-11 The Trustees Of The University Of Pennsylvania Methods and compositions for the treatment of defects in lipoprotein metabolism
US6174527B1 (en) 1995-02-24 2001-01-16 The Trustees Of The University Of Pennsylvania Methods and compositions for gene therapy for the treatment of defects in lipoprotein metabolism
WO1996029423A1 (fr) * 1995-03-20 1996-09-26 Baylor College Of Medicine Compositions et methodes pour induire une infection par des vecteurs retroviraux en dehors de leur gamme d'hotes
US6270996B1 (en) 1995-06-05 2001-08-07 The Trustees Of The University Of Pennsylvania Recombinant adenovirus and adeno-associated virus, cell lines and methods of production and use thereof
US6261551B1 (en) 1995-06-05 2001-07-17 The Trustees Of The University Of Pennsylvania Recombinant adenovirus and adeno-associated virus, cell lines, and methods of production and use thereof
US5756283A (en) * 1995-06-05 1998-05-26 The Trustees Of The University Of Pennsylvania Method for improved production of recombinant adeno-associated viruses for gene therapy
US7105346B2 (en) 1995-06-15 2006-09-12 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US7052881B2 (en) 1995-06-15 2006-05-30 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
WO1997018317A1 (fr) * 1995-11-13 1997-05-22 Commissariat A L'energie Atomique Complexe proteique dodecaedrique adenoviral, composition le contenant et ses applications
FR2741087A1 (fr) * 1995-11-13 1997-05-16 Commissariat Energie Atomique Complexe proteique dodecaedrique adenoviral, procede de preparation, composition le contenant et ses applications
US6083720A (en) * 1995-11-13 2000-07-04 Chroboczek; Jadwiga Dodecahedral adenoviral protein complex, composition containing same and uses thereof
US6649407B2 (en) 1995-11-28 2003-11-18 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6057155A (en) * 1995-11-28 2000-05-02 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US5965541A (en) * 1995-11-28 1999-10-12 Genvec, Inc. Vectors and methods for gene transfer to cells
US6329190B1 (en) 1995-11-28 2001-12-11 Genvec, Inc. Targetting adenovirus with use of constrained peptide motifs
US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
FR2747681A1 (fr) * 1996-04-18 1997-10-24 Commissariat Energie Atomique Complexe proteique dodecaedrique adenoviral, composition le contenant et ses applications
US7232899B2 (en) 1996-09-25 2007-06-19 The Scripps Research Institute Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use
US6569677B1 (en) 1997-04-02 2003-05-27 Transgene S.A. Modified adenoviral fiber and target adenoviruses
US7256036B2 (en) 1997-04-02 2007-08-14 Transgene Modified adenoviral fiber and target adenoviruses
WO1998044121A1 (fr) * 1997-04-02 1998-10-08 Transgene S.A. Fibre adenovirale modifiee et adenovirus cibles
FR2761689A1 (fr) * 1997-04-02 1998-10-09 Transgene Sa Fibre adenovirale modifiee et adenovirus cibles
FR2761688A1 (fr) * 1997-04-02 1998-10-09 Transgene Sa Fibre adenovirale modifiee et adenovirus cibles
US6251677B1 (en) 1997-08-25 2001-06-26 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
WO1999023237A1 (fr) * 1997-11-03 1999-05-14 Commissariat A L'energie Atomique Vecteur peptidique de transfection, composition le contenant et leurs applications
US6750058B1 (en) 1997-11-03 2004-06-15 Commissariat A L'energie Atomique Transfecting peptide vector, composition containing same and applications
US6599737B1 (en) 1998-04-30 2003-07-29 Cornell Research Foundation, Inc. Adenoviral vectors with tandem fiber proteins
US7749493B2 (en) 1998-07-08 2010-07-06 Crucell Holland B.V. Chimeric adenoviruses
US6455314B1 (en) 1998-09-11 2002-09-24 Genvec, Inc. Alternatively targeted adenovirus
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
WO2000034780A3 (fr) * 1998-12-04 2000-10-19 Novartis Ag METHODES ET COMPOSITIONS UTILES POUR CIBLER LE RECEPTEUR αvβ3 ACTIVE PAR LA VITRONECTINE
US6387368B1 (en) 1999-02-08 2002-05-14 The Trustees Of The University Of Pennsylvania Hybrid adenovirus-AAV virus and methods of use thereof
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7250293B2 (en) 1999-05-18 2007-07-31 Crucell Holland B.V. Complementing cell lines
US7270811B2 (en) 1999-05-18 2007-09-18 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7906113B2 (en) 1999-05-18 2011-03-15 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US6372208B1 (en) 1999-09-28 2002-04-16 The Trustees Of The University Of Pennsylvania Method of reducing an immune response to a recombinant virus
WO2002024934A1 (fr) * 2000-09-22 2002-03-28 Consorzio Interuniversitario Per Le Biotecnologie Vecteurs chimeres et leur utilisation pour le transfert de genes heterologues
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
US7344883B2 (en) 2000-11-15 2008-03-18 Crucell Holland B.V. Complementing cell lines
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US9228205B2 (en) 2000-11-15 2016-01-05 Crucell Holland B.V. Complementing cell lines
US7468181B2 (en) 2002-04-25 2008-12-23 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
US7820440B2 (en) 2002-04-25 2010-10-26 Crucell Holland B.V. Means and methods for producing adenovirus vectors
US7611868B2 (en) 2003-05-14 2009-11-03 Instituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Recombinant modified adenovirus fiber protein
WO2011014794A1 (fr) 2009-07-31 2011-02-03 Paxvax, Inc. Vecteurs à base adénovirale
US8865182B2 (en) 2009-07-31 2014-10-21 Paxvax, Inc. Adenoviral-based vectors
WO2017039311A1 (fr) * 2015-08-31 2017-03-09 한양대학교 산학협력단 Composition pour l'administration intracellulaire contenant un peptide dérivé de la protéine vi des adénovirus et composition pharmaceutique contre le cancer contenant ce dernier
US10722587B2 (en) 2015-08-31 2020-07-28 Genemedicine Co., Ltd. Composition for intracellular delivery containing adenovirus protein VI-derived peptide and anticancer pharmaceutical composition containing same

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