- 2014-12-01 (x)
- 1924-10-20 (x)
- Davis, Pamela B. (x)
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Show moreBackground of the invention: The epithelium is the first line of defense against a variety of pathogens. Epithelial cells produce low molecular weight antimicrobial peptides, antibacterial enzymes, and antiproteases. Optimal methods of specifically targeting therapeutic molecules to epithelial cells have been lacking in the art. There is a continuing need in the art for methods of providing therapeutic agents to respiratory epithelia cells in diseases such as cyptic fibrosis, asthma, and emphysema, and to intestinal epithelial cells, for example, in inflammatory bowel diseases. Summary of the invention: It is an object of the invention to provide bifunctional molecules useful for delivery of therapeutic molecules and methods for delivering therapeutic molecules to cells. These and other objects of the invention are provided by one or more embodiments as described below. In one embodiment the invention provides a fusion protein. The fusion protein comprises a single chain Fv molecule directed against a human transcytotic receptor covalently linked to a therapeutic protein. The therapeutic protein may be, for example, .alpha..sub.1 -antitrypsin, a cytokine, such as interleukin-2 or interleukin-10, or a peptide antibiotic. Suitable peptide antibiotics include aerosporin, amphomycin, aspartocin, bacitracins, caperomycins, colistins, dactinomycins, glumamycins, gramicidin D, gramicidin S, mikamycin B, polymixins, pristinamycin, siomycin, staphylomycin S, thiostrepton, tyrocidines, tyrothricin, valinomycin, vancomycin, veramycin B. Any therapeutic protein which one wants delivered to epithelial cells may be used. The fusion protein may further comprise a linker region of less than 50, 40, 30, 20, or 10 amino acid residues. The linker can be covalently linked to and between the single chain Fv molecule and the therapeutic protein. Also provided according to another aspect of the invention is a method of delivering a therapeutic protein to an epithelial cell. The method comprises: administering a fusion protein as described above to a patient, whereby the therapeutic protein is delivered to an epithelial cell. The epithelial cell may be an airway epithelial cell or an intestinal lumen cell, for example. The liver may also be targeted. The administration mode may be any known in the art. However, inhalation and intravenous administration have been found to be both convenient and efficient.Nucleic acid molecules are also provided by the present invention. These encode a fusion protein comprising a single chain Fv molecule directed against a transcytotic receptor covalently linked to a therapeutic protein. The therapeutic protein may be, for example, .alpha..sub.1 -antitrypsin, a cytokine, such as interleukin-2 or interleukin-10, or a peptide antibiotic. Any therapeutic protein which one wants delivered to epithelial cells may be used. The fusion protein may further comprise a linker region of less than 50, 40, 30, 20, or 10 amino acid residues.
http://www.google.com/patents?vid=USPAT6261787
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Show moreTechnical field of the invention: This invention is related to the field of cystic fibrosis. More particularly, it is related to the area of therapeutic treatments and drug discovery for treating cystic fibrosis. Background of the invention: Defects in CFTR, a chloride channel located in the apical membrane of epithelial cells, are associated with the common genetic disease, cystic fibrosis (Quinton, 1986, Welsh and Smith, 1993, Zielenski and Tsui, 1995). CFTR is a 1480 amino acid protein that is a member of the ATP binding cassette (ABC) transporter family (Riordan et al., 1989, Higgins, 1992). Each half of CFTR contains a transmembrane domain and a nucleotide binding fold (NBF), and the two halves are connected by a regulatory, or R domain. The R domain is unique to CFTR and contains several consensus PKA phosphorylation sites (Cheng et al., 1991, Picciotto et al., 1992). Opening of the CFTR channel is controlled by PKA phosphorylation of serine residues in the R domain (Tabcharani et al., 1991, Bear et al., 1992) and ATP binding and hydrolysis at the NBFs (Anderson et al., 1991, Gunderson and Kopito, 1995). Phosphorylation adds negative charges to the R domain, and introduces global conformational changes reflected by the reduction in the .alpha.-helical content of the R domain protein (Dulhanty and Riordan, 1994). Thus, electrostatic and/or allosteric changes mediated by phosphorylation are likely to be responsible for interactions between the R domain and other CFTR domains that regulate channel function (Rich et al., 1993, Gadsby and Naim, 1994). Rich et al., 1991 showed that deletion of amino acids 708-835 from the R domain (.DELTA.R-CFTR), which removes most of the PKA consensus sites, renders the CFTR channel PKA independent, but the open probability of .DELTA.R-CFTR is one-third that of the wild type channel and does not increase upon PKA phosphorylation (Ma et al., 1997, Winter and Welsh, 1997). Thus, it is possible that deletion of the R domain removes both inhibitory and stimulatory effects conferred by the R domain on CFTR chloride channel function. This conclusion is supported by studies that show that addition of exogenous unphosphorylated R domain protein (amino acids 588-858) to wt-CFTR blocks the chloride channel (Ma et al., 1996), suggesting that the unphosphorylated R domain is inhibitory. Conversely, exogenous phosphorylated R domain protein (amino acids 588-855 or 645-834) stimulated the .DELTA.R-CFTR channel, suggesting that the phosphorylated R domain is stimulatory (Ma et al., 1997, Winter and Welsh, 1997). Therefore, it appears that the manifest activity (stimulatory or inhibitory) depends on the phosphorylation state of the R domain. About 25% of the known 700 mutations in CFTR produce a mutant CFTR protein which is properly transported to the apical membrane of epithelial cells but have only low level, residual channel activity.
http://www.google.com/patents?vid=USPAT6770739
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Show moreBackground of the invention: The epithelium is the first line of defense of the airways against invading pathogens. Many of the non-specific defenses against such invaders arise from respiratory epithelial cells. Epithelial cells produce low molecular weight antimicrobial peptides, antibacterial enzymes, and antiproteases. In addition, secretory immunoglobulin A, a non-specific immunoglobulin defense, is trafficked into the airway via a specialized receptor, the polymeric immunoglobulin receptor (pIgR), that is expressed only on epithelial cells. These epithelial defenses are breached early in the life of patients with cystic fibrosis (CF). Once live bacteria reach their surface, the epithelial cells direct the initial inflammatory response by releasing interleukin-8 (IL-8) and interleukin-6 (IL6) as well as reducing expression of interleukin-10 (IL-10). The chemoattractants, combined with increased expression of adhesin molecules for neutrophils, enhance inflammatory cell migration into the airways. Once there, the neutrophils, in an attempt to clear the bacteria, release lytic enzymes in the process. If the neutrophils remain adherent to the epithelium, these enzymes are released right at the epithelial surface. Both mechanical disruption of cells and even low concentrations of neutrophil elastase (NE) result in the greater release of pro-inflammatory mediators from the respiratory epithelium. Thus, the inflammatory response is further enhanced. Several strategies to interrupt this cycle have been proposed. Augmenting the antibacterial defenses of the airway at the epithelial surface may be useful. Prevention of the escalation of the inflammatory responses engendered by the neutrophils migrating into the airway could be accomplished by preventing the action of elastase at the airway cell surface. Both antibiotics and antiproteases are available for clinical use. Unfortunately, the results of clinical studies examining the use of the antiprotease in patients with CF have been disappointing. The systemic administration of alpha.sub.1 -antitrypsin (A.sub.1 AT) is inefficient, and the levels achieved by the intravenous administration of the antiprotease are insufficient to inhibit the overwhelming amount NE in the lung of patients with CF. Aerosolized A.sub.1 AT should permit the direct delivery to the airways, but the antiprotease delivered by nebulization has been uneven and deposits the drug atop the mucus blanket rather than the critical site at the surface of the cell. Thus there is a need in the art for methods to circumvent these difficulties and protect the respiratory epithelial cell surface. Summary of the invention: It is an object of the invention to provide a fusion protein useful for protein delivery. It is another object of the invention to provide a method of delivering a therapeutic protein to an epithelial cell.
http://www.google.com/patents?vid=USPAT6072041
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Show moreBackground of the invention: The epithelium is the first line of defense of the airways against invading pathogens. Many of the non-specific defenses against such invaders arise from respiratory epithelial cells. Epithelial cells produce low molecular weight antimicrobial peptides, antibacterial enzymes, and antiproteases. In addition, secretory immunoglobulin A, a non-specific immunoglobulin defense, is trafficked into the airway via a specialized receptor, the polymeric immunoglobulin receptor (pIgR), that is expressed only on epithelial cells. These epithelial defenses are breached early in the life of patients with cystic fibrosis (CF). Once live bacteria reach their surface, the epithelial cells direct the initial inflammatory response by releasing interleukin-8 (IL-8) and interleukin-6 (IL-6) as well as reducing expression of interleukin-10 (IL-10). The chemoattractants, combined with increased expression of adhesin molecules for neutrophils, enhance inflammatory cell migration into the airways. Once there, the neutrophils, in an attempt to clear the bacteria, release lytic enzymes in the process. If the neutrophils remain adherent to the epithelium, these enzymes are released right at the epithelial surface. Both mechanical disruption of cells and even low concentrations of neutrophil elastase (NE) result in the greater release of pro-inflammatory mediators from the respiratory epithelium. Thus, the inflammatory response is further enhanced. Several strategies to interrupt this cycle have been proposed. Augmenting the antibacterial defenses of the airway at the epithelial surface may be useful. Prevention of the escalation of the inflammatory responses engendered by the neutrophils migrating into the airway could be accomplished by preventing the action of elastase at the airway cell surface. Both antibiotics and antiproteases are available for clinical use. Unfortunately, the results of clinical studies examining the use of the antiprotease in patients with CF have been disappointing. The systemic administration of alpha.sub.1 -antitrypsin (A.sub.1 AT) is inefficient, and the levels achieved by the intravenous administration of the antiprotease are insufficient to inhibit the overwhelming amount NE in the lung of patients with CF. Aerosolized A.sub.1 AT should permit the direct delivery to the airways, but the antiprotease delivered by nebulization has been uneven and deposits the drug atop the mucus blanket rather than the critical site at the surface of the cell. Thus there is a need in the art for methods to circumvent these difficulties and protect the respiratory epithelial cell surface. Summary of the invention: It is an object of the invention to provide a fusion-protein useful for protein delivery. It is another object of the invention to provide a method of delivering a therapeutic protein to an epithelial cell.
http://www.google.com/patents?vid=USPAT6287817
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Show moreField of the invention: The present invention relates to the in vivo delivery of exogenous nucleic acids to cells of multicellular organisms. In particular, the present invention relates to the delivery of exogenous nucleic acids to cells having a serpin enzyme complex receptor on their surface. Background: Functional exogenous genes can be introduced to mammalian cells in vitro by a variety of physical methods, including transfection, direct microinjection, electroporation, and coprecipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes to cells within intact animals. Receptor-Mediated Uncompacted DNA Delivery In VivoReceptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo. This procedure involves linking the DNA to a polycationic protein (usually poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times. The overall level of expression of the transgene in the target tissue is dependent on several factors: the stability of the DNA-carrier complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier ligand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells. Wu, et al., U.S. Pat. No. 5,166,320, discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyornithine, histone, avidin, or protamine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests "asialoglycoprotein (galactose-terminal) ligands".Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) and U.S. Pat. No. 5,354,844 disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis. Wagner, et al., teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event. Direct Injection Of Naked, Uncompacted DNA: The possibility of detecting gene expression by directly injecting naked DNA into animal tissues was demonstrated first by Dubenski et al., Proc. Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmid DNA injected into the liver or spleen of mice was expressed at detectable levels. The DNA was precipitated using calcium phosphate and injected together with hyaluronidase and collagenase. The transfected gene was shown to replicate in the liver of the host animal.
http://www.google.com/patents?vid=USPAT5972901
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Show moreField of the invention: The present invention relates to the in vivo delivery of exogenous nucleic acids to cells of multicellular organisms. In particular, the present invention relates to the delivery of exogenous nucleic acids to cells having a serpin enzyme complex receptor on their surface. Background: Functional exogenous genes can be introduced to mammalian cells in vitro by a variety of physical methods, including transfection, direct microinjection, electroporation, and coprecipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes to cells within intact animals. Receptor-Mediated Uncompacted DNA Delivery in VivoReceptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo. This procedure involves linking the DNA to a polycationic protein (usually poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times. The overall level of expression of the transgene in the target tissue is dependent on several factors: the stability of the DNA-carrier complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier ligand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells. Wu, et al., U.S. Pat. No. 5,166,320, discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyomithine, histone, avidin, or protamine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests "asialoglycoprotein (galactose-terminal) ligands".Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) and U.S. Pat. No. 5,354,844 disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis. Wagner, et al., teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event. Direct Injection of Naked, Uncompacted DNA: The possibility of detecting gene expression by directly injecting naked DNA into animal tissues was demonstrated first by Dubenski et al, Proc. Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmid DNA injected into the liver or spleen of mice was expressed at detectable levels. The DNA was precipitated using calcium phosphate and injected together with hyaluronidase and collagenase. The transfected gene was shown to replicate in the liver of the host animal.
http://www.google.com/patents?vid=USPAT6200801
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