+

WO1996030536A1 - Apport d'un acide nucleique cible egf - Google Patents

Apport d'un acide nucleique cible egf Download PDF

Info

Publication number
WO1996030536A1
WO1996030536A1 PCT/US1996/004017 US9604017W WO9630536A1 WO 1996030536 A1 WO1996030536 A1 WO 1996030536A1 US 9604017 W US9604017 W US 9604017W WO 9630536 A1 WO9630536 A1 WO 9630536A1
Authority
WO
WIPO (PCT)
Prior art keywords
complex
nucleic acid
egf
gene
cell
Prior art date
Application number
PCT/US1996/004017
Other languages
English (en)
Inventor
Richard J. Cristiano
Jack A. Roth
Original Assignee
Board Of Regents, The University Of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Priority to AU53706/96A priority Critical patent/AU5370696A/en
Publication of WO1996030536A1 publication Critical patent/WO1996030536A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the present invention relates generally to the field of gene delivery.
  • the invention relates to receptor-mediated gene targeting to lung cancer cells.
  • the invention relates to epidermal growth factor (EGF) -mediated nucleic acid delivery and expression in lung cell lines.
  • EGF epidermal growth factor
  • Lung cancer is one of the most frequently diagnosed malignant neoplasms throughout the world (Hammar, 1988) .
  • lung cancer is now the leading cause of cancer death, killing more than 140,000 people annually.
  • Age-adjusted mortality from lung cancer has recently surpassed that for breast cancer in women.
  • Implementation of smoking-reduction programs has decreased the prevalence of smoking, but lung cancer mortality rates are expected to remain high well into the 21st century.
  • lung cancer therapy Although considerable research effort has been devoted to lung cancer therapy, the overall prognosis is poor, with present treatments such as surgery, radiotherapy and chemotherapeutics having a relatively limited effect (Hammar, 1988) .
  • Important considerations in effective therapy of lung cancer include a high selectivity in targeting cancer cells but not normal cells, i.e., elevated levels of expression of a receptor on tumor cell surfaces, and efficient delivery of complete gene sequences into these target cells.
  • Recent advances in recombinant DNA and gene delivery technologies suggest that gene therapy may be a promising alternative to standard radio- and chemotherapeutic regimens.
  • the present vectors now available for gene delivery into lung cancer cells include recombinant retroviruses and adenoviruses. Each has been shown to work in vivo, but these vectors have limitations, such as the lack of cell-specific targeting, gene size limitations and safety concerns.
  • receptor-mediated gene targeting takes advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993) . Furthermore, in comparison with viral delivery systems (Morgan and Anderson, 1993) , receptor-mediated gene delivery allows greater flexibility of DNA size and sequence because the DNA to be delivered does not need to be packaged into viral capsids. This helps avoid tedious clonal selection and virus-production processes. These characteristics make the system an attractive prospect for cancer therapy.
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • a cell receptor-specific ligand Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid
  • ASOR (Wu and Wu, 1987) and transferrin (Wagner et al . , 1990) .
  • transferrin (Wagner et al . , 1990) .
  • ASOR Seru and Wu, 1987
  • transferrin a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al . , 1993; Perales et al . , 1994) .
  • ASOR and transferrin receptors are very different. Transferrin receptors are found in many different cell types, while ASOR receptors are almost exclusively distributed on the sinusoidal domain of the hepatocytes (Wu and Wu, 1987) .
  • Epidermal growth factor has also been used to deliver genes to squamous carcinoma cells (Myers, EPO).
  • compositions for use in gene therapy it is an object of the present invention to provide improved compositions for use in gene therapy. It also is an object of the present invention to provide methods for the use of such compositions and, in particular, use in the treatment of cancer.
  • the present invention addresses the need for improved therapy for lung cancer and other diseases by providing compositions and methods for their use.
  • the present invention encompasses a nucleic acid-binding agent/EGF complex that is capable of selectively delivering nucleic acid into cells via an EGF receptor uptake system.
  • the present invention also provides compositions and methods to promote the uptake and expression of the nucleic acids in cells by use of an endosomal lysis agent.
  • a complex comprising an epidermal growth factor (EGF) receptor- binding peptide and a nucleic acid-binding agent.
  • EGF epidermal growth factor
  • the peptide is whole EGF, a fragment of EGF or a chemically synthesized portion of EGF.
  • the nucleic acid-binding agent is a polycationic moiety.
  • the nucleic acid-binding agent is polylysine.
  • the invention includes an endosomal lysis agent such as an infectious, replication-deficient adenovirus.
  • Complexes according to the present invention may further comprise a nucleic acid.
  • the nucleic acid may be at least of a length selected from the group consisting of 10, 20, 50, 100, 500, 1000, 5000 and 10,000 base pairs.
  • the nucleic acid encodes a therapeutic gene.
  • the therapeutic gene is operably linked to a promoter that is active in a cell expressing an EGF receptor.
  • the therapeutic gene may be a tumor suppressor such as p53 or pl6.
  • the therapeutic gene may encode cystic fibrosis transmembrane conductance regulator (CFTR) , blood clotting factor IX or other disease ameliorating proteins.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the nucleic acid may encode an antisense construct or a ribozyme.
  • the antisense construct or ribozyme may target an oncogene transcript.
  • the antisense construct or ribozyme targets a ras oncogene.
  • the invention includes a pharmaceutical composition comprising a complex as described above and a pharmaceutically-acceptable carrier, diluent or excipient.
  • a kit comprising an EGF receptor- binding peptide and a nucleic acid-binding agent.
  • the ligand and nucleic acid-binding agent are complexed.
  • the kit may further comprise a nucleic acid encoding a therapeutic gene.
  • the ligand, nucleic acid-binding agent and nucleic acid are complexed.
  • Another embodiment of the invention includes a method of delivering a gene to a cell expressing EGF receptor.
  • This method comprises providing a EGF- containing complex for the delivery of a gene to a cell and contacting said complex with said cell.
  • a method of delivering a gene to a cell comprises contacting the cell with an endosomal lysis agent.
  • the endosomal lysis agent is an infectious, replication-deficient adenovirus.
  • Another embodiment includes a method of treating a mammal with a lung tumor in vivo comprising providing a pharmaceutical composition as described above and administering the pharmaceutical composition to the mammal.
  • Preferred embodiments of this method include the method where the cells of the lung tumor overexpress EGF.
  • inventions include a method of treating a mammal with a lung tumor in vivo, wherein said administering is selected from the group consisting of intravenous injection, subcutaneous injection, direct perfusion, inhalation, intratracheal injection and intra-peritoneal injection.
  • FIG. 1 Diagrammatic representation of the
  • EGF/PLL/DNA complex in which EGF/Biotin (B) is linked to poly-L-lysine (PLL) by streptavidin.
  • PLL poly-L-lysine
  • FIG. 2 Diagrammatic representation of the EGF/PLL/DNA complex when it is coupled to the replication defective adenovirus that has been modified by the covalent attachment of PLL to the viral capsid. Coupling occurs between the positive charge of the lysine (K) groups and the negative charge of the DNA, resulting in the EGF/DNA/Adenovirus complex.
  • K lysine
  • FIG. 3 Adenovirus enhancement of DNA delivery through the EGF/PLL/DNA complex, by using a non-modified adenovirus.
  • the numbers of adenoviral particles per cell were 0, 30, 100, 300, 1000, 3000, and 10,000.
  • Three micrograms of pCMV/3-Gal DNA in complex form with the EGF/PLL conjugate were incubated with the non-modified adenovirus.
  • the percentage blue staining represents the percentage of cells staining positive for /3-Gal expression.
  • FIG. 4 Adenovirus enhancement of DNA delivery through the EGF/PLL/DNA complex, by direct attachment of the complex to the E5 adenovirus .
  • the numbers of adenoviral particles per cell were 0, 30, 100, 300, 1000, 3000, and 10,000.
  • the amount of DNA incubated with the cells is equal to 0.000001 ng/adenoviral particle.
  • the percentage blue staining represents the percentage of cells staining positive for 3-Gal expression.
  • FIG. 5 The quantitation of jS-Gal expression in lung cancer cells after incubation with the EGF/PLL/DNA complex.
  • the samples incubated with the cells are as follows: 3 ⁇ g EGF/PLL/DNA complex + adenovirus (Filled bar) ; EGF/PLL/DNA complex + E5 adenovirus (Open bar) .
  • 3 adenoviral particles/cell was used.
  • the o-nitrophenol /3-galactopyranoside analysis was done 24 h after the initial incubation with the cells.
  • a unit is defined as nmol of o-nitrophenol formed per minute (Nielson et al . , 1983) .
  • FIG. 6 Analysis of DNA delivery into various lung cancer cell lines by the EGF/PLL/DNA complex.
  • the cell lines were incubated with the following: 3 ⁇ g EGF/PLL/DNA complex + adenovirus (Filled bar) ; EGF/PLL/DNA complex + E5 adenovirus (Open bar) .
  • 3 adenoviral particles/cell was used. Histologic staining was done 24 h after administration of the samples to the cells.
  • the present invention relies on the interaction between a specific receptor-ligand pair, namely, EGF and its cognate receptor.
  • EGF peptide the peptide will direct the nucleic acid to cells expressing the receptor and initiate its uptake by the cell .
  • the attachment of the nucleic acid to EGF is not direct, however, but is facilitated by a nucleic acid-binding moiety that is, in turn, bound to EGF.
  • complex is meant to include one or more molecules bound together by any physical or chemical means and encompasses the terms “conjugate” and “couple.”
  • conjugate and “couple.”
  • the complex may be tightly or weakly bound together in a highly specific or totally non-specific way.
  • ligand is meant to include any molecule that binds to another molecule or macromolecule, the latter being a "receptor.”
  • nucleic acid- binding agent is intended to refer to any molecule or compound that binds a nucleic acid.
  • a ligand and nucleic acid-binding agent may be complexed by any means including: covalent, ionic, hydrophobic, intercalative, van der Waals' or a combination of these forces and others.
  • the targeting ligand is epidermal growth factor (EGF) or another EGF receptor-binding peptide.
  • EGF is a single-chain polypeptide of 53 residues with three disulfide bonds which define three looped regions 1-20, 14-31, and 32-53 (Savage et al . , 1972) . It has been suggested that residues in positions 20-31 contain a major receptor binding region ( Komoriya et al . , 1984) and that deletion of the COOH-terminal five or six residues leads to a marked reduction in receptor affinity (Cohen and Carpenter, 1975; Hollenberg and Gregory 1980) . Due to its high degree of cross-species similarity, it is envisioned that any EGF receptor-binding protein from any mammalian source can be employed according to the present invention.
  • EGF The cognate receptor for EGF is over-expressed on the surface of lung tumor cell lines (Putnam et al . , 1992) .
  • a number of other tumor and normal cells also express an EGF receptor.
  • EGF receptor-binding peptides to lung tumor cells, it is proposed that the invention is generally applicable to situations where an EGF peptide recognizes a receptor.
  • cancer cell types such as breast cancer, glioblastoma and squamous carcinoma are mentioned by way of example.
  • normal tissue such as epithelium or muscle also provides a suitable target.
  • EGF peptides need to retain some significant receptor binding activity in order to function according to the present invention. It is a routine matter to test for such binding with purified receptor in vi tro by assays such as gel retardation assays, filter binding techniques, affinity chromatography and precipitation or sedimentation methods.
  • an in situ approach could be used. For example, one could label the peptide to be tested and mix the peptide with cells expressing the receptor on their cell surface under conditions which permit binding. Subsequently, this mixture is submitted to treatment that separates the unbound peptide from bound peptide. The amount of peptide bound to cells can be determined and compared with a control samples comprising (i) peptide with no specific binding affinity for EGF receptor and (ii) cells lacking an EGF receptor. Further binding studies may be performed, including the use of EGF as a competitor, to compare affinities for receptor.
  • functional output can be used to determine binding and cellular uptake of a complex via an EGF receptor.
  • a nucleic acid encoding a reporter gene can be conjugated to the EGF ligand, as discussed further below.
  • the reporter gene is expressed and the reporter product's activity monitored.
  • One such reporter gene is ⁇ -galactosidase, as described in the disclosed example.
  • Nucleic acid-binding agents include proteins, polypeptides, peptides, antibodies, nucleotides, carbohydrates, fatty acids, organic or inorganic compounds or a combination of these and others. Nucleic acid-binding agents may bind to single-stranded or double-stranded DNA, to single-stranded or double- stranded RNA, by chemical or physical forces or by a combination of the two.
  • a nucleic acid-binding agent may (i) have affinity only for the nucleic acid itself, (ii) have affinity for both the nucleic acid and another molecule, thereby forming a bridge between the two or (iii) have indirect affinity for the nucleic acid via affinity for another molecule that has affinity for the nucleic acid.
  • the coupling of a nucleic acid-binding agent and EGF ligand must occur in such a manner that does not interfere with the binding of the EGF ligand to EGF receptor.
  • internalization of the EGF ligand complex via normal receptor-mediated endocytosis also is retained.
  • this recognition and internalization delivers the nucleic acid into a target cell in a form suitable for the expression or for interaction with target endogenous nucleic acid.
  • the nucleic acid-binding agent may insert itself between base pairs of double-stranded nucleic acids in an intercalative manner or bind in the minor or major groves of double-stranded nucleic acids. This binding may be sequence specific or completely unrelated to sequence.
  • nucleic acids may be cross-linked with other molecules with chemically of photochemically reactive groups.
  • the nucleic acid-binding agent covalently links the nucleic acid to another molecule.
  • the nucleic acid- binding agent is one of the coupling agent as described below such as carbodiimide.
  • covalent coupling of the nucleic acid may alter its specificity and preclude proper gene expression or target nucleic acid recognition.
  • linear or single stranded nucleic acid may be a requirement for covalent coupling of the nucleic acid to an EGF receptor.
  • nucleic acids are negatively charged molecules which means that they may be repelled from cell surfaces, making transfer difficult via the endosomal lysis pathway. Therefore a size and type restriction may be necessary for the efficient delivery of nucleic acid directly bound to an EGF protein or fragment.
  • a preferred embodiment of the invention is a polycationic moiety that depends on electrostatic-dominated binding involving sequence-neutral interactions between the cationic groups and the negatively charged phosphates on nucleic acid.
  • the polycationic moiety binds DNA strongly resulting in the formation of a toroid complex where the negative charge of nucleic acid molecule is completely neutralized. This soluble toroid complex may be internalized via normal receptor-mediated endocytosis.
  • Any type of nucleic acid may be used, from single stranded mRNA to double stranded circular plasmids.
  • any size of nucleic acid may be used, as long as there is a source of negative charge for the polycationic moiety to bind.
  • these polycationic moieties may include a natural polyamine such as spermine and spermidine.
  • the polycationic moiety may be an artificially produced moiety, such as polylysine.
  • nucleic acid-binding agent In order for the invention to function properly, certain criteria with regard to the nucleic acid-binding agent need to be fulfilled.
  • the nucleic acid to be delivered into the cell must bind to the nucleic acid- binding agent without loosing its integrity in any way.
  • the complex comprising of ligand, nucleic acid- binding agent and nucleic acid must be in soluble form to allow greater accessibility of the complex to cells in vi tro and in vivo.
  • the nucleic acid Once the complex is internalized within the host cell, the nucleic acid must have access to its target sequence while avoiding degradation.
  • the complex includes an EGF moiety and a nucleic acid-binding moiety.
  • the two moieties are bound to each other via a linking entity.
  • a linking entity is streptavidin (or avidin) and biotin.
  • the streptavidin (or avidin) -biotin interaction is the strongest known non-covalent biological interaction between a protein and ligand (Pierce, 1994) .
  • the linking entity may include agents such as carbodiimides, N-succinimidyl, 3- (2-pyridyldithio) propionate, succinimmidyl,4- (N- maleimidomethyl) cyclohexane-1-carboxylate, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines.
  • agents such as carbodiimides, N-succinimidyl, 3- (2-pyridyldithio) propionate, succinimmidyl,4- (N- maleimidomethyl) cyclohexane-1-carboxylate, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines.
  • the present invention also includes an endosomal lysis agent that serves to increase release of the complex from endosomes once internalized by the cell but prior to fusion with a lysosome, thereby preventing degradation of the nucleic acid and/or the complex.
  • An endosomal lysis agent is any agent that causes a disruption of the endosome sufficient to allow release of a complex without undue disruption of other cellular components.
  • adenoviral particles are an affective endosomal lysis agent. The particles may be mixed with the complex or coupled to the complex, as described in Example 1. In preferred embodiments, modified adenoviruses are used to overcome potential safety problems.
  • an infectious, replication-deficient adenovirus particle (Cotten et al . , 1992) or adenovirus with reduced toxicity (Cotten et al . , 1993) may be used.
  • Other viruses, modified or unmodified, may be used in the present invention. These include, but are not limited to, human papilloma virus (HPV) or adeno-associated virus (AAV) .
  • fusogenic viral peptides can be used in lieu of whole virus particles.
  • influenza hemagglutinin HA-2 terminal peptides have been demonstrated to augment gene transfer efficiency greater than 100-fold by the receptor-mediated gene transfer into HeLa cells (Wagner et al . , 1992; Plank et al . , 1994) .
  • recent studies in protein trafficking and endosomal physiology have identified several endogenous proteins associated with vesicle budding, membrane fusion (Rothman and Orci, 1992) and protein translocation (G ⁇ rlich and Rapoport, 1993) . These proteins can be used to reduce the lysosomal degradation of transgenes in receptor-mediated gene delivery.
  • Toxins such as ricin, cholera enterotoxin, endotoxin, pertussis toxin, diphtheria toxin or fragments thereof may be employed as endosomal lysis agents .
  • Toxins often have a high affinity for cell surfaces whereby they are introduced into the cell via the endosomal lysis system and then released.
  • ricin binds to galactose
  • diphtheria toxin binds to ganglioside GM1, on cell surfaces.
  • toxins are often harmful, if not lethal, to the cell.
  • any modified, or fragment of, toxin that maintains the ability to enter the cell and be released from the endosome without undue damage to the cell may be used in the present invention.
  • the primary use for the complexes described above is in the delivery of genes to cells. Such delivery may be accomplished in vi tro, as in laboratory procedures for transforming cells lines, or in vivo, as in the treatment of disease states for which a there is a genetic basis. Thus, it is proposed that the present invention is generally applicable to any situation where one desires high level expression of a recombinant protein in a target or host cell through the use of the invention.
  • compositions and methods described herein will be useful in standard laboratory procedures with a variety of different gene constructs.
  • standard expression cassettes including a gene of interest can be transferred to a target cell for transient expression.
  • Other vectors that permit stable transformation, either chromosomal or episomal, may be used.
  • therapeutic gene is intended to refer to any foreign nucleic acid introduced into a cell for the potential benefit of the cell or to an organism containing that cell. This process may include the introduction of: (i) a normal allele of a gene into a cell that either does not express its own copy of the gene or has a defective copy;
  • toxic genes such as cytokines, to interfere with the expression of oncogenes or viral genes and thus inhibit neoplastic cell growth or viral replication; or
  • antisense constructs and ribozymes that act on disease-related nucleic acid targets in cells.
  • a particularly useful gene is a tumor suppressor.
  • tumor suppressors There are numerous tumor suppressors well know to those in the art, preferred examples including p53, pl6, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC and MCC. This list is not intended to be exhaustive of the various tumor suppressors known in the art but, rather, is exemplary of the more common tumor suppressors.
  • a third modality for gene therapy involves the synthesis of antimicrobial agents within cells susceptible to infection by the corresponding viruses.
  • the therapeutic gene could be any product that serves to block, reduce or inhibit viral replication and or damage to host cells.
  • the therapeutic gene is stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non ⁇ specific location (gene augmentation) . In further embodiments, the gene may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.
  • the therapeutic gene would comprise complementary DNA (cDNA) .
  • cDNA complementary DNA
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • the cDNA is operably linked to a promoter.
  • the promoter is active in one or more cell types expressing an EGF protein receptor and, more preferably, in a lung cell.
  • a "promoter” refers to a DNA sequence recognized by the synthetic components of the cell, required to initiate the specific transcription of a gene. What is meant by the phrase “operably linked” is that the promoter is in the correct location and orientation in relation to the gene to allow the promoter to facilitate expression of the gene.
  • the particular promoter that is employed to control the expression of the therapeutic gene is not believed to be important, so long as it is capable of expressing the therapeutic gene in the targeted cell.
  • a human cell it is preferable to position the therapeutic gene coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the cytomegalovirus (CMV) promoter is a preferred promoter.
  • CMV cytomegalovirus
  • promoters derived from RSV, N2A, LN, or SV40 Preferred embodiments of the invention would include lung-specific promoters such as the Clara cell 10 kDa promoter (CC 10 ) (Stripp et al . , 1992) , phosphoglycol kinase (PGK) , or lung epithelial cell-specific surfactant protein B (SPB) (Bohinski et al . , 1994) .
  • CC 10 Clara cell 10 kDa promoter
  • PGK phosphoglycol kinase
  • SPB lung epithelial cell-specific surfactant protein B
  • the therapeutic gene may be designed to alleviate a non-cancerous disease state.
  • cystic fibrosis CF
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Delivery of the CFTR gene into the lungs of affected individuals may prove to be an important therapy for CF.
  • genetic therapy for hemophilia could be achieved by introduction of a therapeutic gene encoding a clotting factor into muscle cells.
  • antisense molecules may be employed.
  • the term "antisense nucleic acid” is intended to refer to the targeting of oligonucleotides against complementary base sequences in DNA and RNA. Extracellular oligonucleotides enter the cell and specifically bind their targets, interfering with transcription, RNA processing and transport or translation. Targeting double-stranded (ds) DNA with oligonucleotide leads to triple-helix formation; targeting RNA will lead to double-helix formation.
  • the antisense DNA or RNA that is introduced will be complementary to a selected cellular gene, such as an oncogene sequence or some other sequence whose expression one seeks to diminish through antisense application.
  • any sequence of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence.
  • shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will be used.
  • the antisense constructs of the present invention will down-regulate oncogene expression.
  • the construct will inhibit or suppress the ultimate expression of the target gene, presumably by binding to the target RNA or DNA, and thereby preventing its translation or transcription, respectively.
  • the most preferred oncogenes for application of the present invention will be those which specifically exist in lung cells or any other cell that expresses an EGF protein receptor on its surface.
  • oncogenes and oncogene families such as ras, myc, myb, mos, met, neu, raf, erb, arc, fps, fms, jun and a l. Again, this list is not intended to be exhaustive but, rather, is exemplary of the more common oncogenes.
  • the antisense construct may correspond to a transcript for an organism such as Streptococcus pneumoniae , Haemophilus influenzae, Klebsiella pneumoniae, Mycoplasma pij.euino.niae, rhinovirus or coronavirus.
  • targeted ribozymes may be used.
  • ribozyme is refers to the an RNA-based enzyme capable targeting cleaving particular base sequences in DNA and RNA.
  • Ribozymes can either be targeted directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as DNA encoding the desired ribozymal RNA. Ribozymes may be used and applied in much the same way as described for antisense nucleic acids.
  • EGF receptor-binding peptide changes may be made in the structure of EGF receptor-binding peptide while maintaining the desirable receptor-binding characteristics.
  • certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites of ligands such as an EGF receptor-binding peptide. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
  • arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
  • hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8) ; cysteine/cystine (+2.5) ; methionine (+1.9) ; alanine (+1.8); glycine (-0.4) ; threonine (-0.7) ; serine (-0.8) ; tryptophan (-0.9) ; tyrosine (-1.3) ; proline (-1.6) ; histidine (-3.2) ; glutamate (-3.5) ; glutamine (-3.5) ; aspartate (-3.5) ; asparagine (-3.5); lysine (-3.9); and arginine (-4.5) .
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein.
  • the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0) ; lysine (+3.0) ; aspartate (+3.0 ⁇ 1) ; glutamate (+3.0 ⁇ 1) ; serine (+0.3) ; asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • peptidyl compounds described herein may be formulated to mimic the key portions of the peptide structure.
  • Such compounds which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents.
  • the generation of a structural functional equivalent may be achieved by the techniques of modelling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • EGF receptor-binding peptide/nucleic acid-binding agent it will be necessary to prepare the complex as a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells.
  • compositions of the present invention comprise an effective amount of the nucleic acid, such as a therapeutic gene, bound to an EGF receptor-binding peptide/nucleic acid binding agent, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • a pharmaceutically acceptable carrier or aqueous medium Such compositions can also be referred to as inocula.
  • therapeutic gene is intended to refer to any nucleic acid introduced into a cell for the potential benefit of the cell or the individual organism as a whole. This includes, among other things, as obvious to one skilled in the art, tumor suppressors, cytokines, antisense constructs and ribozymes.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • other pharmaceutically acceptable forms include, e . g. : inhalents and such like, that administer the active ingredients by aerolization; tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions and even mouthwashes.
  • the active compounds also may be formulated for parenteral administration, e. g. , formulated for injection via the intravenous, intramuscular, subcutaneous, intratracheal or even intraperitoneal routes.
  • parenteral administration e. g. , formulated for injection via the intravenous, intramuscular, subcutaneous, intratracheal or even intraperitoneal routes.
  • the preparation of an aqueous composition that contains the therapeutic gene complex as an active ingredient will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or a-queous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • a therapeutic gene complexed to an EGF receptor- binding peptide/nucleic acid-binding agent of the present invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, 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, histidine, procaine and the like.
  • Therapeutic formulations in accordance with the present invention may also be reconstituted in the form of inhalants which may contain EGF receptor-binding peptide/nucleic acid binding agent and therapeutic gene complex alone, or in conjunction with other agents, such as, e . g. , pentamidine.
  • the carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus ny additional desired ingredient from a previously sterile-filtered solution thereof.
  • DMSO dimethyl methacrylate
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580) .
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • kits comprising an EGF receptor-binding peptide and a nucleic acid-binding agent form another aspect of the invention.
  • Such kits will generally contain, in suitable container means, pharmaceutically- acceptable formulation of the complex comprising of EGF receptor-binding peptide and a nucleic acid-binding agent.
  • a nucleic acid of choice also included in the kit may a nucleic acid of choice and a pharmaceutically-acceptable formulation of an endosomal lysis agent.
  • the kit may have a single container means that contains EGF receptor-binding peptide and nucleic acid agent, with or without the nucleic acid of choice and the endosomal lysis agent, or it may have distinct container means for each compound.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the EGF receptor-binding peptide/nucleic acid agent may also be formulated into a syringeable composition.
  • the container means may itself be an inhalent, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, or even applied to and mixed with the other components of the kit.
  • the components of the kit may also be provided in dried or lyophiiized forms.
  • reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
  • the container means generally will include at least one vial, test tube, flask, bottle, syringe or other container means, into which an EGF receptor-binding peptide/nucleic acid agent/nucleic acid complex may be placed,
  • the kit also will generally contain a second vial or other container into which the endosomal lysis agent may be placed.
  • the kits also may comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e . g. , injection or blow-molded plastic containers into which the desired vials are retained.
  • kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • an instrument may be an inhalent, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • an EGF peptide/nucleic acid-binding agent complex In order for an EGF peptide/nucleic acid-binding agent complex to deliver nucleic acid to a cell, it must first contact the cell.
  • the phrase "contact,” as used herein, is intended to refer to the structural proximity of two or more moieties needed before functional interaction can occur.
  • functional interaction may be defined as the recognition of the complex by EGF receptor on the target cell surface and subsequent internalization of the complex by the cell.
  • EGF peptide/nucleic acid- binding agent complex In order to contact an EGF peptide/nucleic acid- binding agent complex with a cell in vi tro, it is a simple matter to add or admix the complex with the cells, as evidenced in Example 1.
  • contacting the target cell or tissue could involve direct injection or inhalation. Alternatively, it could involve the administration of the soluble EGF complex into the blood stream. Size may play a role in successful delivery and contact of the complex to the relevant cells.
  • Previous study of an ASOR- polylysine-DNA complex prepared under similar procedures gave molecular size of 80-100 nm (Cristiano et al . , 1993). Perales et al . (1994) have recently reported that by changing salt conditions, it is possible to modulate the sizes of galactosylated poly(L-lysine) /DNA complex (to about 10 nm) . The formation of such small complexes was found to correlate»with the prolonged expression of transgene in the livers of intact animals.
  • Another in vi tro use of the present invention is to determine whether cells or cell lines, express functional EGF receptor on their surface. This can be evaluated by admixing the EGF ligand/nucleic acid-binding agent complexed with a reporter gene with the cells to be evaluated, as described in Example 1. Where the reporter gene is - ⁇ -galactosidase, cells expressing or overexpressing EGF receptor will produce clearly visible, blue-stained cells. This procedure may be used for diagnosing cancer cells as many tumor cells overexpress the EGF receptor. This would be particularly valuable for lung, breast and glioblastoma cells.
  • EGF Human EGF
  • PLL poly-L-lysine
  • Streptavidin (Pierce Chemical Co.) was modified with the use of N-Succinimidyl-3- (2-pyridyldithio) -propionate (Pierce Chemical Co.) to contain one sulfhydryl group (Carlsson et al . , 1972) .
  • Poly-L-lysine was modified to contain one sulfhydryl group by using Traut's reagent
  • the modified streptavidin and modified PLL were then combined and allowed to incubate overnight at room temperature under argon, to allow for disulfide bond formation.
  • the resulting streptavidin/PLL was then purified by using a Mono Q column (Pharmacia Fine Chemicals, Piscataway, NJ.) .
  • the final sample contained streptavidin linked to PLL in a one-to-one ratio.
  • the third step involved incubation of the EGF/biotin with the streptavidin/PLL for 30 min. at room temperature.
  • the DNA/protein complexes were made by adding increasing molar ratios of the EGF/PLL conjugate in 150 ⁇ l of HBS to 6 ⁇ g of DNA in 350 ⁇ l of HBS, vortexing the sample and then incubating for 30 min. at room temperature. The resulting samples were either analyzed by agarose gel electrophoresis to determine charge neutralization on the DNA sample or added to cells for uptake studies.
  • the plasmid pCMV/0-Gal which contains the E. coli beta-galactosidase (3-Gal) gene under the control of the cytomegalovirus enhancer and promoter, was used for cell transduction studies. All plasmid DNA preparations were purified by using the Mega Prep plasmid preparation kit (Qiagen, Studio City, Ca) .
  • a diagrammatic representation of the EGF/PLL/DNA complex can be seen in FIG. 1.
  • the ADV (E5) was then either dialyzed against 2 M NaCl and then stored at - 20°C in 10% glycerol or stored in 10% glycerol (Cristiano et al . , 1993) .
  • a diagrammatic representation of the EGF/PLL/DNA complex coupled to adenovirus can be seen in FIG. 2.
  • the cell lines used in the analysis were SW620, a colon adenocarcinoma (Murphy et al . , 1990); H460a (Putnam et al . , 1992) and H1299 (Mitsudomi et al . , 1992) , large cell lung carcinomas; H322 (Mitsudomi et al . , 1992) , H226b (Putnam et al . , 1992) , and H226Br (Fujiwara et al . , 1994), squamous lung carcinomas; H596 (Mitsudomi et al .
  • EGF/PLL conjugate was capable of binding DNA
  • increasing molar amounts of the EGF/PLL conjugate were incubated with a set amount of the plasmid pCMV/ / 3-Gal.
  • the samples were then analyzed by gel electrophoresis on a 1% agarose gel. Correct binding of the EGF/PLL conjugate with the DNA should result in the charge on the DNA being neutralized, causing it to be retained in the well of the gel. This charge neutralization has been shown to contribute to efficient DNA delivery into cells (Cristiano et al . , 1993) .
  • the charge on the DNA did not neutralize.
  • the amount of the EGF/PLL conjugate was increased, a greater proportion of the DNA was rendered neutral, with the DNA being completely neutralized at a molar ratio of EGF/PLL to DNA of 150/1.
  • the EGF/PLL/DNA complex that showed complete charge neutralization was then incubated with three different cell lines; SW620, H460a, and H1299.
  • the cell line SW620 does not express EGF receptors at the cell surface (Murphy et al . , 1990).
  • the cell line H460a has been determined to have 6xl0 4 EGF receptors per cell and a higher affinity for EGF (Putnam et al . , 1992).
  • the status of the EGF receptor on the cell line H1299 has not been determined.
  • H460a has a mutation in the K-ras gene and H1299 has a deletion of the p53 gene.
  • adenovirus enhanced the delivery of the EGF/PLL/DNA complex into the cell through endosomal lysis
  • 3 ⁇ g of the EGF/PLL/DNA complex was added to the cells along with an increasing amount of the adenovirus (FIG. 3) .
  • a viral titer of 10 2 viral particles/cell there was a slight increase in the percentage of H460a and H1299 cells staining positive.
  • the maximum number of positive-staining cells for these cell lines occurred at a concentration of 3xl0 3 adenoviral particles/cell, whereas the cell line SW620 showed very low numbers of cells staining positive for ⁇ - Gal activity at all adenoviral titers used.
  • the cells were incubated with 3 ⁇ g of DNA in complex form along with 10 3 adenoviral particles/cell and with a 200- fold molar excess of free EGF.
  • the percentage of positive staining cells was reduced from 65% to 12% for the H460 cell line and reduced from 35% to 8% for the H1299 cell line, indicating specific delivery through the EGF receptor.
  • competition was done with a 200-fold molar excess of insulin, no reduction in positive staining cells occurred.
  • EGF/PLL/DNA complex can be delivered efficiently into lung cancer cell lines along with the help of the replication-defective adenovirus, a more suitable complex for DNA delivery in vi tro and in vivo would be the direct coupling of the adenovirus to the complex. This has also been shown to be effective with other DNA/protein complexes (Cristiano et al . , 1993).
  • a virus that was modified to have a large amount of PLL attached to the viral capsid was generated.
  • This modified adenovirus (E5) has been shown to have a large proportion of targeting occur through the coupled ligand and its receptor (Cristiano et al . , 1993) .
  • E5 modified adenovirus
  • FIG. 4 When DNA was delivered by coupling the EGF/PLL/DNA complex to E5, the efficiency of DNA delivery into the cells was enhanced (FIG. 4) .
  • the titer of the adenovirus necessary to achieve maximal transduction was between 3xl0 2 and 10 3 adenoviral particles/cell, almost an order of magnitude less than was needed when the complex was simply administered along with the unmodified adenovirus. This was seen by levels of transduction in the cell lines H460a and H1299 (FIG. 4) .
  • the uptake of the EGF/DNA/E5 complex by the cell line SW620 was also increased (up to 15% transduction) , possibly indicating that the EGF/PLL/DNA complex has a greater ability to enter the cell through the adenoviral receptor when it is linked to the adenovirus.
  • the S-Gal expression was -quantitated.
  • 3 ⁇ g of pCMV//3-Gal in complex form was incubated with the cells along with 10 3 adenoviral particles/cell, the levels of / S-Gal expression in the cell lines H1299 and H460a ranged from 0.7 to 0.9 units of / S-Gal/5xl0 5 cells, whereas no / ⁇ -Gal was detected in the cell line SW620 (FIG. 5) , which is permissive for adenovirus infection. This indicates that the DNA/protein complex was being taken up through the EGF receptor and not through the adenovirus receptor.
  • the level of / S-Gal expression was greatly increased, with up to 4.4 units of / 8-Gal/5xl0 5 cells expressed (FIG. 5) .
  • the cell line SW620 also showed an increase in /S-Gal expression when the modified adenovirus was used, again indicating that a small percentage of the DNA was being delivered through the adenovirus receptor, but at much lower levels than the amount that entered through the EGF receptor.
  • the ability of the EGF/PLL/DNA complexes to deliver DNA into several different lung cancer cell lines was assessed.
  • the complex, along with the adenovirus (either non-modified or modified) was incubated with the cells at a viral titer of 10 3 adenoviral particles/cell (FIG. 6) .
  • the levels of transduction ranged from 15% (H226b) to 70% (H460a) (FIG. 6) .
  • the E5 adenovirus was used along with the EGF/PLL/DNA complex, the percentage of cells transduced ranged from 15% (H226b) to 99% (H1299) (FIG. 6) .
  • the coupling of the adenovirus to the EGF/PLL/DNA complex enhanced the delivery of the DNA into the cells.
  • the EGF/PLL/DNA complex allowed the efficient delivery of DNA into several lung cancer cell lines.
  • the results shown here agree with other reports in which the attachment of DNA to protein/PLL conjugates resulted in a receptor-dependent process for uptake that could be enhanced by an endosomal lysis agent.
  • the uptake of the EGF/PLL/DNA complex occurred specifically through the EGF receptor, which was shown by the lack of DNA uptake into the cell line SW620 and by competition with free EGF, when analyzed by either histologic staining or quantitation of / 8-Gal expression.
  • the delivery of DNA by the complex was enhanced by adenovirus, which acts as an endosomal lysis agent.
  • the efficiency of delivery was further enhanced by coupling the complex directly to the adenovirus as seen by the increase in the number of cells transduced when the E5 modified adenovirus was used.
  • the delivery of DNA by the EGF/DNA/E5 complex could be competed with excess free EGF, but this was not as successful as when the complex and the adenovirus were separate. This may be due to the following factors; 1) the interaction of the adenovirus with its receptor, as seen by an increase in the number of cells staining positive for the cell line SW620, 2) a change in the interaction between the EGF and its receptor, as there are more EGF molecules per EGF/DNA/E5 complex, 3) the larger number of DNA molecules that are being delivered by the complex, contributing to higher levels of expression being achieved, or 4) a combination of the above factors.
  • compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Bohinski et al The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol . Cell . Biol . 1 (9) :5671-5681 (1994) .
  • Biotinylated epidermal growth factor a useful tool for the histochemical analysis of specific binding sites. Histochem. J. 22:426-432 (1990) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention décrit un complexe induit par des récepteurs qui apporte sélectivement de l'acide nucléique dans des cellules. Le peptide liant le facteur de croissance épidermique (EGF) au récepteur agit en tant que ligand de ciblage et il est complexé avec un composant qui fixe l'acide nucléique. Un gène marqueur conjugué à un agent de liaison peptide EGF/acide nucléique a été transféré avec succès dans un nombre de lignées cellulaires différentes qui expriment des taux élevés de récepteur.
PCT/US1996/004017 1995-03-24 1996-03-25 Apport d'un acide nucleique cible egf WO1996030536A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU53706/96A AU5370696A (en) 1995-03-24 1996-03-25 Egf-targeted nucleic acid delivery

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41052695A 1995-03-24 1995-03-24
US08/410,526 1995-03-24

Publications (1)

Publication Number Publication Date
WO1996030536A1 true WO1996030536A1 (fr) 1996-10-03

Family

ID=23625107

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/004017 WO1996030536A1 (fr) 1995-03-24 1996-03-25 Apport d'un acide nucleique cible egf

Country Status (2)

Country Link
AU (1) AU5370696A (fr)
WO (1) WO1996030536A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028432A1 (fr) * 1996-12-20 1998-07-02 Pepsyn Limited Procede d'augmentation du taux de transfection des cellules
US6693083B2 (en) 1997-08-05 2004-02-17 Watson Pharmaceuticals, Inc. Conjugates targeted to the interleukin-2 receptor
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US8106098B2 (en) 1999-08-09 2012-01-31 The General Hospital Corporation Protein conjugates with a water-soluble biocompatible, biodegradable polymer
US8247383B2 (en) * 1999-08-09 2012-08-21 The General Hospital Corporation Drug-carrier complexes and methods of use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0273085A1 (fr) * 1986-12-29 1988-07-06 IntraCel Corporation Procédé pour faire entrer des acides nucléiques dans des cellules eucaryotes
WO1991014696A1 (fr) * 1990-03-29 1991-10-03 Gilead Sciences, Inc. Conjugues a base de disulfure d'oligonucleotide et d'un agent de transport

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0273085A1 (fr) * 1986-12-29 1988-07-06 IntraCel Corporation Procédé pour faire entrer des acides nucléiques dans des cellules eucaryotes
WO1991014696A1 (fr) * 1990-03-29 1991-10-03 Gilead Sciences, Inc. Conjugues a base de disulfure d'oligonucleotide et d'un agent de transport

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J.CHEN ET AL.: "A novel gene delivery system using EGF receptor-mediated endocytosis", FEBS LETTERS, vol. 338, no. 2, 31 January 1994 (1994-01-31), AMSTERDAM NL, pages 167 - 169, XP002009636 *
K.ZATLOUKAL ET AL.: "Receptor-mediated cytokine delivery to tumor cells for generation of cancer vaccines. Keystone symposium on cellular immunity and the immunotherapy of cancer, Taos New Mexico, USA March 17-24, 1993", JOURNAL OF CELLULAR BIOCHEMISTRY SUPPLEMENT 17D, 1993, pages 129, XP002009637 *
R.J.CHRISTIANO AND J.A.ROTH: "Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidermal growth factor receptor", CANCER GENE THERAPY, vol. 3, no. 1, January 1996 (1996-01-01), pages 4 - 10, XP000577163 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028432A1 (fr) * 1996-12-20 1998-07-02 Pepsyn Limited Procede d'augmentation du taux de transfection des cellules
US6159737A (en) * 1996-12-20 2000-12-12 Smith; John Arthur Method of enhancing the rate of transfection of cells
US6693083B2 (en) 1997-08-05 2004-02-17 Watson Pharmaceuticals, Inc. Conjugates targeted to the interleukin-2 receptor
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US8106098B2 (en) 1999-08-09 2012-01-31 The General Hospital Corporation Protein conjugates with a water-soluble biocompatible, biodegradable polymer
US8247383B2 (en) * 1999-08-09 2012-08-21 The General Hospital Corporation Drug-carrier complexes and methods of use thereof

Also Published As

Publication number Publication date
AU5370696A (en) 1996-10-16

Similar Documents

Publication Publication Date Title
Dohmen et al. Nanosized multifunctional polyplexes for receptor-mediated siRNA delivery
Pichon et al. Histidine-rich peptides and polymers for nucleic acids delivery
US6846809B2 (en) PEI: DNA vector formulations for in vitro and in vivo gene delivery
US20110306656A1 (en) Biologically tolerated low molecular weight polyethylenimines
JPH09508100A (ja) 核酸を含む組成物、調製および使用
JPH08504565A (ja) 高分子のトランスロケーションシグナル促進核輸送
CN113811611A (zh) 无毒cas9酶及其应用
US6339139B1 (en) Receptor-mediated gene transfer system for targeting tumor gene therapy
US5766899A (en) Targeted nucleic acid delivery into liver cells
JP2004528266A (ja) 薬物送達用組成物
JP2002316997A (ja) 目的とするアニオン性物質を細胞に導入するための複合体
US20090011984A1 (en) Biotin-binding receptor molecules
WO2009053937A2 (fr) Supports à base de nano-lipide pour une administration ciblée de vecteurs viraux et leur procédé de fabrication
US7081495B1 (en) Cationic compounds and their use as macro molecular carriers
van Rossenberg et al. Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery
Saraswat et al. DNA as therapeutics; an update
US20020155157A1 (en) Compositions and methods for polynucleotide delivery
WO1996030536A1 (fr) Apport d'un acide nucleique cible egf
JP2003503569A (ja) 核酸を細胞に輸送するためのコポリマー
EP1489093A1 (fr) Peptides, compositions medicinales contenant ce peptide et compositions medicinales anticancereuses
Dai et al. Construction of an EGF receptor-mediated histone H1 0-based gene delivery system
WO2001008710A1 (fr) Expression de gene au moyen de polynucleotides modifies de maniere covalente
RU2537262C2 (ru) Молекулярные конъюгаты с поликатионным участком и лигандом для доставки в клетку и ядро клетки днк и рнк
US20030125239A1 (en) Compositions for drug delivery
US20040019188A1 (en) Targeting through integrins

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载