WO2000039145A1 - Transfert genique cible utilisant des recepteurs couples a une proteine g - Google Patents

Transfert genique cible utilisant des recepteurs couples a une proteine g Download PDF

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WO2000039145A1
WO2000039145A1 PCT/US1999/030658 US9930658W WO0039145A1 WO 2000039145 A1 WO2000039145 A1 WO 2000039145A1 US 9930658 W US9930658 W US 9930658W WO 0039145 A1 WO0039145 A1 WO 0039145A1
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Prior art keywords
conjugate
ligand
group
vector
vectors
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PCT/US1999/030658
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English (en)
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Richard C. Boucher, Jr.
Raymond J. Pickles
Janet L. Rideout
William Pendergast
Benjamin R. Yerxa
James G. Douglass, Iii
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The University Of North Carolina At Chapel Hill
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Priority to JP2000591056A priority Critical patent/JP2002533110A/ja
Priority to AU22088/00A priority patent/AU2208800A/en
Priority to CA002355227A priority patent/CA2355227A1/fr
Priority to EP99966577A priority patent/EP1140967A1/fr
Publication of WO2000039145A1 publication Critical patent/WO2000039145A1/fr

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • 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
    • 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
    • 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
    • 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
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors

Definitions

  • This invention relates to methods and systems useful in the transfer of nucleic acids into eukaryotic cells.
  • the capacities to introduce a particular foreign or native gene sequence into a mammalian cell and to control the expression of that gene are of substantial value in the fields of medical and biological research. Such capacities provide a means for studying gene regulation, and for designing a therapeutic basis for the treatment of disease.
  • the introduction of a particular foreign or native gene into mammalian host cells is facilitated first by introducing a gene sequence into a suitable nucleic acid vector.
  • a variety of methods have been developed which are capable of permitting the introduction of such a recombinant vector into a desired host cell.
  • the use of viral vectors can result in the rapid introduction of the recombinant molecule in a wide variety of host cells.
  • viral vectors have been employed in order to increase the efficiency of introducing a recombinant nucleic acid vector into host cells.
  • Viruses that have been employed as vectors for the transduction and expression of exogenous genes in mammalian cells include SV40 virus (see, e.g., H. Okayama et al., Molec. Cell. Biol. 5, 1136-1142 (1985)); bovine papilloma virus (see, e.g., D. DiMaio et al., Proc. Natl. Acad. Sci. USA 79, 4030-4034 (1982)); adenovirus (see, e.g., J.E. Morin et al., Proc. Natl.
  • AAV adeno-associated virus
  • herpes simplex virus see, e.g., A.I. Geller, et al., Science 241, 1667 (1988)
  • retroviral vectors include a relatively restricted host range, based in part on the level of expression of the membrane protein that serves as the viral receptor. M.P. Kavanaugh et al., Proc. Natl. Acad. Sci USA 91, 7071-7075 (1994).
  • the invention is based upon the unexpected discovery by the present inventors that the rate limiting step of viral vector uptake by cell surface receptors is not, as originally thought, the binding event of the virus to the receptor, but rather the intemalization of the receptor itself. Accordingly, this invention relates to new complexes that facilitate the transfer of nucleic acids into eukaryotic cells. This invention allows for targeting transfer vectors to specific cell types, attachment of the vectors to the cells and regulated cellular intemalization of the vectors.
  • This invention comprises binding a transfer vector to a receptor that is internalized by a cell.
  • the receptor is one that is either internalized by a cell upon the cell's exposure to a specific ligand, or for which a receptor may be induced to internalize by exposure to such a ligand.
  • Figure 1 is a schematic drawing illustrating a virus-receptor complex of the present invention. This Figure illustrates an adenovirus targeted to an internalizing seven transmembrane receptor.
  • Figure 2 is a schematic representation of particular embodiments of the present invention.
  • Figure 3 is a graphical representation of the Cl " secretory responses of human airway epithelia to lumenal NECA (A 2b agonist), isoproterenol, bradykinin, or ATP (all lO ⁇ M).
  • Figure 4 A is a graphical representation of the dose-effect relationship between bs-Ab concentration and gene transfer efficiency in A9-null cells ( • ) compared with HA-P2Y 2 -A9 administered sequentially (O) or as preformed conjugates (A).
  • Figure 4B is a graphical representation of a study to evaluate the specificity of increased gene transfer with bs-Ab in A9-HA-P2Y and A9-null cells pre-treated with specific or non-specific bs-Ab or after chronic desensitization of HA-P2Y 2 receptors by pretreatment with ATP ⁇ S.
  • Figure 5 is a graphical representation of gene transfer with bs-Ab in null A9 and A9 cells expressing an HA-tagged BK ⁇ receptor.
  • Figure 6 is a graphical representation of gene transfer in CHO cells with bs-Ab to
  • HA-tagged P2Y 2 and ⁇ 2 receptors and adenovirus fiber protein HA-tagged P2Y 2 and ⁇ 2 receptors and adenovirus fiber protein.
  • Figure 7 is a graphical representation of biotin-UTP stimulation of inositol phosphate formation in P2Y receptor expressing ( • ) but not wild-type ( ⁇ ) astrocytoma cells.
  • Figure 8 is a graphical representation of the stimulation of gene transfer in A9
  • Figure 9A is a graphical representation of a comparison of agonist potency of U 2 P 4 and UTP in astrocytoma cells expressing P2Y receptors.
  • Figure 9B is a graphical representation of the metabolic stability of U P 4 compared with UTP in cystic fibrosis sputum.
  • a transfer vector-receptor complex of the present invention comprises a transfer vector bound to a receptor that is capable of being internalized into a cell.
  • the transfer vector may contain an exogenous nucleic acid sequence (e.g., a gene), and may express an exogenous protein or peptide.
  • the transfer vector is targeted to a seven transmembrane (7-TM) receptor by means of an antibody specific to the receptor, by means of a peptide expressed by the transfer vector that specifically binds said receptor, or by means of a natural or modified ligand.
  • the transfer vector may be any suitable vector, including a viral vector, a plasmid, an oligonucleotide, or RNA/DNA chimeric molecules, as is described more fully hereinbelow. Interaction between the 7-TM receptor and the targeted complex results in receptor-complex intemalization, thereby introducing the heterologous nucleic acid carried by the transfer vector into the cell where it is expressed.
  • a complex (i.e., a conjugate) of the present invention comprises a viral vector 10 (which is illustrated in the figure as an adeno vims) attached to a 7-TM receptor 20, which receptor 20 is present on a cell surface 100.
  • the viral vector 10 is attached to the 7-TM receptor 20 by means of a bifunctional bridging antibody 30.
  • the bifunctional bridging antibody 30 is composed of one antibody 40 which specifically binds the viral vector.
  • the antibody 40 is chemically cross-linked to antibody 50, which specifically binds the 7-TM receptor 20.
  • the viral vector 10 is illustrated as an adeno vims vector (AdV), it will be understood that the present invention may also be practiced with other viral vectors, including but not limited to human and nonhuman retro vims (i.e., Maloney vims such as Moloney Murine Leukemia Vims and lentivimses) vectors, adeno-associated vims (AAV) vectors, and herpes vims vectors ( Figure 2).
  • the viral vectors of the present invention may be attenuated vimses or may be rendered non-replicative by any method known to one skilled in the art.
  • the viral vectors of the present invention will have the capacity to include exogenous nucleic acids.
  • the delivery of the heterologous nucleic acid facilitates the replication of the heterologous nucleic acid within the target cell, and the subsequent production of a heterologous protein therein.
  • a heterologous protein is herein defined as a protein or fragment thereof wherein all or a portion of the protein is not naturally expressed by the target cell.
  • a nucleic acid or gene sequence is said to be heterologous if it is not naturally present in the wild type of the viral vector used to deliver the gene into a cell (e.g., the wild-type adenovims genome).
  • nucleic acid sequence or “gene sequence, " as used herein, is intended to refer to a nucleic acid molecule (preferably DNA).
  • Such gene sequences may be derived from a variety of sources including DNA, cDNA, synthetic DNA, RNA or combinations thereof.
  • Such gene sequences may comprise genomic DNA which may or may not include naturally occurring introns.
  • genomic DNA may be obtained in association with promoter sequences or poly-adenylation sequences.
  • the gene sequences of the present invention are preferably cDNA.
  • Genomic or cDNA may be obtained in any number of ways. Genomic DNA can be extracted and purified from suitable cells by means well- known in the art. Alternatively, mRNA can be isolated from a cell and used to prepare cDNA by reverse transcription, or other means.
  • the nucleotide sequence of the inserted heterologous gene sequence or sequences may be of any nucleotide sequence.
  • the inserted heterologous gene sequence may be (or may include) a reporter gene sequence or a selectable marker gene sequence.
  • a reporter gene sequence is any gene sequence which, when expressed, results in the production of a protein whose presence or activity can be monitored. Examples of suitable reporter genes include the gene for galactokinase, beta-galactosidase, chloramphenicol acetyltransferase, beta-lactamase, etc.
  • the reporter gene sequence may be any gene sequence whose expression produces a gene product which affects cell physiology.
  • a selectable marker gene sequence is any gene sequence capable of expressing a protein whose presence permits one to selectively propagate a cell which contains it.
  • selectable marker genes include gene sequences capable of conferring host resistance to antibiotics (e.g., puromycin, ampicillin, tetracycline, kanamycin, and the like), or of conferring host resistance to amino acid analogues, or of permitting the growth of bacteria on additional carbon sources or under otherwise impermissible culture conditions.
  • a gene sequence may be both a reporter gene and a selectable marker gene sequence.
  • the most preferred reporter genes of the present invention are the lacZ gene which encodes the beta-galactosidase activity of E. coli; and the gene encoding puromycin resistance.
  • reporter or selectable marker gene sequences are sufficient to permit the recognition or selection of the vector in normal cells.
  • the reporter gene sequence will encode an enzyme or other protein which is normally absent from mammalian cells, and whose presence can, therefore, definitively establish the presence of the vector in such a cell.
  • the heterologous gene sequence may also comprise the coding sequence of a desired product such as a suitable biologically active protein or polypeptide, immunogenic or antigenic protein or polypeptide, or a therapeutically active protein or polypeptide.
  • a therapeutically active protein or polypeptide e.g., the heterologous gene sequence encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein or biologically active analogs, fragments, or derivatives thereof.
  • the heterologous gene sequence may comprise a sequence complementary to an RNA sequence, such as an antisense RNA sequence, which antisense sequence can be administered to an individual to inhibit expression of a complementary polynucleotide in the cells of the individual. Expression of the heterologous gene may provide immunogenic or antigenic protein or polypeptide to achieve an antibody response, which antibodies can be collected from an animal in a body fluid such as blood, serum or ascites.
  • heterologous gene sequence a gene sequence which already possesses a promoter, initiation sequence, or processing sequence.
  • the transfer vector is non-viral.
  • suitable vectors include, but are not limited to, oligonucleotides (including RNA, DNA, synthetic and modified nucleic acids), plasmids, and RNA/DNA chimeric molecules as described by E. Kometz.
  • Oligonucleotide vectors include antisense oligonucleotides and oligonucleotides that function as ribozymes.
  • the non- viral transfer vectors of the present invention are able to include exogenous nucleic acids as described hereinabove with respect to viral vectors. Oligonucleotides, plasmids, and RNA/DNA chimeric molecules can be synthesized or produced by any suitable method known in the art.
  • Receptors that may be used to carry out the present invention belong to the family of 7-TM receptors. See generally, S. Watson et al., The G-Protein Linked Receptor
  • 7-TM receptors are G protein coupled receptors. Any mammalian G protein coupled receptor, and the nucleic acid sequences encoding these receptors, may be employed in practicing the present invention. Examples of such receptors include, but are not limited to, dopamine receptors, muscarinic cholinergic receptors, ⁇ -adrenergic receptors, opiate receptors, cannabinoid receptors, serotonin receptors, ⁇ -adrenergic receptors, and purinoceptors.
  • the term "receptor" as used herein is intended to encompass subtypes of the named receptors, and mutants and homologs thereof, along with the nucleic acid sequences encoding the same.
  • the 7-TM receptor for use according to the present invention is a purinoceptor as discussed in greater detail below (e.g., P2Yj, P2Y 2 , P2Y 4 , P2Y 6 and P2Y ⁇ ), an adenosine receptor (i.e., Al, A2, and A3, and sub-types thereof), a bradykinin receptor (e.g., BKj and BK ⁇ ), or a ⁇ -adrenergic receptor (e.g., ⁇ i, ⁇ and ⁇ 3 ).
  • C 5A complement receptor More preferred are the P2Y , BK ⁇ , A 2B , ⁇ 2 , and C 5A receptors, with the P2Y receptor being most preferred.
  • ligands that may be used to carry out the present invention include nucleotides, nucleosides, catecholamines (e.g., dopamine, 5-hydroxytryptophan), C5A, and bradykinin(s).
  • the P2Y (also known as the P 2 u)- purinoceptor undergoes intemalization upon activation with ATP, UTP and analogs thereof. These receptor types are abundant in number on the lumenal surface of the human respiratory epithelium. Mason, S. J., et al. 1991. Br. J. Pharmacol. 103 , 1649- 1656. Molecular conjugation of AdV to P2Y 2 - receptors, followed by activation of these receptors by ATP/UTP, leads to intemalization of the vector-ligand-receptor complex into endosomes and thus provide an alternative entry pathway for AdV into the well differentiated (WD) epithelium, and thereafter to gene expression.
  • one strategy for targeting transfer vectors carrying heterologous nucleic acids to 7-TM receptors for intemalization into the cell is with a bispecific bridging antibody.
  • the bispecific antibody is directed against epitopes on both the transfer vector and the 7-TM receptor of interest (i.e., has a combining region that specifically recognizes the transfer vector and a combining region that specifically recognizes the 7-TM receptor), thereby forming a "bridge" between the transfer vector and the receptor. Binding of the bispecific bridging antibody to the 7-TM receptor induces intemalization of the receptor.
  • the transfer vector is preferably a viral vector, more preferably, AdV
  • the bispecific antibody comprises a monoclonal antibody directed against the fiber (knob) protein of the adenovims.
  • antibodies refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG are particularly preferred.
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al, Molec. Immunol. 26, 403-11 (1989).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading U.S. Patent No. 4,474,893, or Cabilly et al., U.S. Patent No. 4,816,567.
  • the antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in SegAl et al., U.S. Patent No. 4,676,980.
  • Antibodies may be polyclonal or monoclonal, with monoclonal being preferred.
  • the antibodies are bridging antibodies that are specific to both the target receptor and the transfer vector.
  • the bridging antibody is preferably a monoclonal antibody directed to the adenovims fiber (knob) protein.
  • monoclonal antibodies and bridging antibodies comprising monoclonal antibodies that are directed to specific epitopes of the 7-TM receptor of interest.
  • Antibodies that bind to the same epitope (i.e., the specific binding site) that is bound by an antibody to the 7-TM receptor can be identified in accordance with known techniques, such as their ability to compete with labeled antibody to the 7-TM receptor in a competitive binding assay.
  • Antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2, and Fc fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques.
  • Polyclonal antibodies used to carry out the present invention may be produced by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen to which a monoclonal antibody to the 7-TM receptor binds, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures.
  • Monoclonal antibodies used to carry out the present invention may be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265, 495-97 (1975).
  • a solution containing the appropriate antigen may be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • the hybridoma cells are then grown in a suitable media and the supernatant screened for monoclonal antibodies having the desired specificity.
  • Monoclonal Fab fragments may be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989).
  • Antibodies specific to the 7-TM (e.g., P2Y ) receptor may also be obtained by phage display techniques known in the art.
  • Antibodies as described herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • Antibodies as described herein may likewise be conjugated to detectable groups such as radiolabels (e.g., S, I, I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., S, I, I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • antigenic equivalents refers to proteins or peptides which bind to an antibody which binds to the protein or peptide with which equivalency is sought to be established. Antibodies which are used to select such antigenic equivalents are referred to as “selection antibodies” herein. E. Non-Antibody Based Targeting Strategies
  • bispecific bridging antibodies as means of targeting the transfer vector to the 7-TM receptor for intemalization.
  • alternative targeting strategies include those utilizing peptides and 7-TM receptor agonist/antagonists.
  • the peptide can be a natural ligand that binds to the 7- TM receptor.
  • Peptide agonists and antagonists of 7-TM receptors are known in the art.
  • novel 7-TM receptor agonists/antagonists can be identified as described by U.S. Patent No. 5,482,835 to King et al.
  • the peptide can be identified by phage display techniques, or any other method in the art, as binding to the 7-TM receptor.
  • nucleic acids encoding the peptide are fused to or inserted into the gene encoding the AdV knob protein, such that a knob-peptide chimeric protein is expressed. It is known, for example, that exogenous nucleic acid can be expressed in the C-terminus or the HI loop region of the knob protein. In alternate embodiments, concatamers of the peptide are expressed in the knob protein.
  • a receptorbody is a truncated receptor in which a peptide that binds to a 7-TM receptor is substituted for the intramembrane and intracellular region of the adenoviral receptor.
  • the external portion of the viral receptors acts as an "antibody” to the binding region of the vims (it replaces the need for an antibody to AdV).
  • the external domain oof the Adv receptor can be fused to a peptide ligand for a 7-TM receptor.
  • This complex will bind to a recombinant AdV so displaying bradykinin peptide on the surface of the vims.
  • the peptide ligand fused to the tmncated AdV receptor will then target this complex to the peptide ligand' s cognate receptor on the cell surface.
  • the transfer vector can be targeted to the 7-TM receptor by a chemically-linked high affinity agonist/antagonist of the 7-TM receptor.
  • High-affinity agonists/antagonists may be peptide ligands, as described above or are other molecules such as nucleotides (e.g., ATP, UTP) , dinucleotides (described in more detail hereinbelow), and derivatives thereof.
  • P2Y receptor ligands, particularly P2Y 2 receptor ligands, that can be used to carry out the present invention include all of the compounds, particularly the nucleotides and dinucleotides that are P2Y2 ligands and are disclosed in W.
  • X], X 2 and X 3 are each independently either O “ or S " ; preferably, X 2 and X 3 are O " ;
  • Rj is O, imido, methylene or dihalomethylene (e.g., dichloromethylene or difluoromethylene); preferably, R ⁇ is oxygen or difluoromethylene;
  • R is H or Br; preferably, R 2 is H; particularly preferred compounds of Formula I are uridine 5'-triphosphate (UTP) and uridine 5'-O-(3-thiotriphosphate) (UTP ⁇ S).
  • a dinucleotide is depicted by the general Formula II:
  • B and B' are each independently a purine residue or a pyrimidine residue linked through the 9- or 1- position, respectively;
  • Z OH or N 3 ;
  • Y' H or OH; provided that when Z is N 3 , Y is H or when Z' is N 3 , Y' is H.
  • the furanose sugar is preferably in the ⁇ -configuration.
  • the furanose sugar is most preferably in the ⁇ -D-configuration.
  • Preferred compounds of Formula II are the compounds of Formula Ila:
  • B' is defined in Formulas lie and lid;
  • B' is defined in Formulas lie and lid;
  • X CH 2 , CF 2 , or NH; n and m-1;
  • B and B' are defined in Formulas lie and lid .
  • Another preferred group of the compounds of Formula II are the compounds of Formula lib or the pharmaceutically acceptable salts thereof:
  • B and B' are uracil, attached at N-l position to the ribosyl moiety, then the total of m + n may equal 3 or 4 when X is oxygen.
  • the ribosyl moieties are in the D- configuration, as shown, but may be L-, or D- and L-. The D- configuration is preferred.
  • the substituted derivatives of adenine include adenine 1 -oxide; l,N6-(4- or 5- substituted etheno) adenine; 6-substituted adenine; or 8-substituted aminoadenine, where R' of the 6- or 8-HNR' groups are chosen from among: arylalkyl (C ⁇ 6 ) groups with the aryl moiety optionally functionalized as described below; alkyl; and alkyl groups with functional groups therein, such as: ([6-aminohexyl] carbamoylmethyl)-, and ⁇ -acylated-amino(hydroxy, thiol and carboxy) derivatives where the acyl group is chosen from among, but not limited to, acetyl, trifluroroacetyl, benzoyl, substituted-benzoyl, etc., or the carboxylic moiety is present as its ester or amide derivative, for example, the ethyl
  • B or B' or both in Formula lib may be a pyrimidine with the general formula of Figure lid, linked through the 1 -position:
  • R l is hydroxy, mercapto, amino, cyano, aralkoxy, C ⁇ - alkoxy, Cj -6 alkylamino, and dialkylamino, the alkyl groups optionally linked to form a heterocycle;
  • R 5 is hydrogen, acyl, C ⁇ -6 alkyl, aroyl, C ⁇ -5 alkanoyl, benzoyl, or sulphonate;
  • R ⁇ is hydroxy, mercapto, alkoxy, aralkoxy, C 1-6 -alkylthio, C 1-5 disubstituted amino, triazolyl, alkylamino, or dialkylamino, where the alkyl groups are optionally linked to form a heterocycle or linked to N-3 to form an optionally substituted ring, ureido or substituted ureido wherein the ureido N atom remote from the pyrimidine ring is substituted with a C 1-6 alkyl group, or a C 6- ⁇ 0 aryl group or a C 7- ⁇ 2 arylalkyl group where in turn the alkyl, aryl and arylalkyl moieties may be substituted with amino, hydroxy, thio, carbonyl or sulfonyl substituents which themselves may be substituted.
  • R 7 is hydrogen, hydroxy, cyano, nitro, alkenyl, with the alkenyl moiety optionally linked through oxygen to form a ring optionally substituted on the carbon adjacent to the oxygen with alkyl or aryl groups, substituted alkynyl or hydrogen where R 8 is amino or substituted amino and halogen, alkyl, substituted alkyl, perhalomethyl (e.g., CF 3 ), C -6 alkyl, C 2-3 alkenyl, or substituted ethenyl (e.g., allylamino, bromvinyl and ethyl propenoate, or propenoic acid), C 2-3 alkynyl or substituted alkynyl when Re is other than amino or substituted amino and together R 5 - R ⁇ may form a 5- or 6-membered saturated or unsaturated ring bonded through N or O at R ⁇ , such a ring may contain substituents that themselves contain functionalities;
  • R 8 is hydrogen, alkoxy, arylalkoxy, alkylthio, arylalkylthio, carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, or phenylthio.
  • the dotted lines in the 2- to 6— positions are intended to indicate the presence of single or double bonds in these positions; the relative positions of the double or single bonds being determined by whether the R 4 , R ⁇ , and R 7 substituents are capable of keto-enol tautomerism.
  • the acyl groups advantageously comprise alkanoyl or aroyl groups.
  • the alkyl groups advantageously contain 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms optionally substituted by one or more appropriate substituents, as described below.
  • the aryl groups including the aryl moieties of such groups as aryloxy are preferably phenyl groups optionally substituted by one or more appropriate substituents, as described below.
  • the above mentioned alkenyl and alkynyl groups advantageously contain 2 to 8 carbon atoms, particularly 2 to 6 carbon atoms, e.g., ethenyl or ethynyl, optionally substituted by one or more appropriate substituents as described below.
  • alkyl, alkenyl, alkynyl, and aryl groups are advantageously selected from halogen, hydroxy, C alkoxy, C 1-4 alkyl, C 6- ⁇ 2 arylalkoxy, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic, amino, and substituted amino wherein the amino is singly or doubly substituted by a C ⁇ alkyl, and when doubly substituted, the alkyl groups optionally being linked to form a heterocycle.
  • R 2 is O or is absent; or
  • Ri and R 2 taken together may form optionally substituted 5-membered fused imidazole ring; or Ri of the 6-HNR ⁇ group or R 3 of the 8-HNR 3 group is chosen from the group consisting of:
  • i is hydroxy, mercapto, amino, cyano, aralkoxy, C ⁇ -6 alkylthio, C ⁇ -6 alkoxy, C ⁇ -6 alkylamino or dialkylamino, wherein the alkyl groups of said dialkylamino are optionally linked to form a heterocycle;
  • R 5 is hydrogen, acyl, C 1-6 alkyl, aroyl, C ⁇ -5 alkanoyl, benzoyl, or sulphonate;
  • R 6 is hydroxy, mercapto, alkoxy, aralkoxy, C ⁇ -6 -alkylthio, C ⁇ -5 disubstituted amino, triazolyl, alkylamino or dialkylamino, wherein the alkyl groups of said dialkylamino are optionally linked to form a heterocycle or linked to N 3 to form an optionally substituted ring;
  • R 5 - R ⁇ 5 together forms a 5 or 6-membered saturated or unsaturated ring bonded through N or O at Re, wherein said ring is optionally substituted;
  • R 7 is selected from the group consisting of: (a) hydrogen,
  • alkenyl wherein the alkenyl moiety is optionally linked through oxygen to form a ring optionally substituted with alkyl or aryl groups on the carbon adjacent to the oxygen
  • R 8 is selected from the group consisting of:
  • Ri, Xi, X 2 and X 3 are defined as in Formula I;
  • R 5 and Re are H while R 7 is nothing and there is a double bond between N-3 and C-4 (cytosine), or
  • Ri, Xj, X , and X 3 are defined as in Formula I;
  • R 3 and j are H while R 2 is nothing and there is a double bond between N-1 and C-6 (adenine), or R 3 and j are H while R 2 is O and there is a double bond between N-1 and C-6 (adenine 1 -oxide), or
  • Formulas I, II, III, and IV herein illustrate the active compounds in the naturally occurring D-configuration, but the present invention also encompasses compounds in the L-configuration, and mixtures of compounds in the D- and L-configurations, unless otherwise specified. The naturally occurring D-configuration is preferred.
  • Some compounds of Formulas I, II, III, and IV can be made by methods which are well known to those skilled in the art and in accordance with known procedures (Zamecnik, P., et al, Proc. Natl Acad.
  • high affinity agonists or antagonists can be directly linked to the transfer vector using sulfo-N-hydroxysuccinimide ( ⁇ HS) as described in more detail below.
  • ⁇ HS sulfo-N-hydroxysuccinimide
  • Examples of compounds that can be used to carry out the present invention include compounds of Formula I-IV above, and include compounds having the general formula:
  • X may be O or S
  • A is a purine or pyrimidine base (e.g., adenine, guanine, thymine, cytosine, uracil)(each purine or pyrimidine base is preferably joined to the ribose or deoxyribose ring by covalent bond to the 9 nitrogen in the case of purines, or by covalent bond to the 1 nitrogen in the case of pyrimidines);
  • a purine or pyrimidine base e.g., adenine, guanine, thymine, cytosine, uracil
  • Ri is H or OH; and n is from 1 to 4 or 6, preferably 2, 3 or 4.
  • the transfer vector is covalently or noncovalently joined or conjugated to the purine or pyrimidine base, or the corresponding ribose or deoxyribose ring (e.g., of the compounds of Formula I-IV above), or attached to the terminal phosphate moiety of compounds represented by Formulae I, II and IV above, by any suitable means, such as by covalently joining a linking chain (e.g., a linking polymer chain) thereto in any suitable position (e.g., a ring carbon such as the 5 carbon in a pyrimidine, or the 2, 6 or 8 carbon in a purine), to which linking group the ligand may be covalently attached, or to which linking group a biotin group may be attached, with a biotin group covalently joined to the ligand (see below) and the two biotin groups joined to one another by means of an avidin group to which both biotin groups are joined or conjugated.
  • a linking chain e.g., a linking polymer
  • X may be O or S
  • a and B are each independently a purine or pyrimidine base (e.g., adenine, guanine, thymine, cytosine, uracil); preferably, one of A or B is uracil; in one particularly preferred embodiment, A is uracil and B is cytosine;
  • Ri and R 2 are each independently selected from the group consisting of H or OH; n is from 1 to 4 or 6, preferably 2, 3 or 4; and said transfer vector is covalently or noncovalently conjugated or joined to A or B or the ribose or deoxyribose ring to which A or B is joined, either directly or indirectly by means of a linking group, in the same manner as described above.
  • a biotin (B) -UTP is used as a targeting agonist for the P2Y 2 receptor.
  • the B-UTP can interact with a biotinylated viral transfer vector in the presence of streptavidin (SA) to give a vims-biotin-S A — biotin-UTP complex that will be targeted to the P2Y receptor.
  • SA streptavidin
  • oligonucleotides, plasmids, and RNA/DNA chimeric molecules can be synthesized or produced to incorporate B-UTP or any other suitable labeled nucleotide.
  • biotinyl moiety is linked to the pyrimidine base by an aminoallyl linker;
  • another particular embodiment of a biotin-dinucleotide conjugate is represented by the formula:
  • biotinyl moiety is linked to the pyrimidine base by a linker attached through alkylation of a thiol group
  • P2Y ligand conjugates may be formed by linking a biotinyl moiety or linking the vector directly through an etheno moiety fused to a purine or pyrimidine base.
  • LINKER represents any straight chain of 2-24 atoms in length, or aromatic (e.g. phenyl, naphthyl), or heterocyclic rings (e.g. benzothiophene, isoxazole, pyridine, piperidine) optionally substituted and covalently linking the etheno moiety to the FUNCTION moiety;
  • FUNCTION represents any of the methods described herein useful to introduce a heterologous nucleic acid into a cell through intemalization of the receptor-ligand complex;
  • X is CH 2 , CC1 2 , CF 2 , NH or O;
  • Y is O or S.
  • etheno-linked conjugates are ethenocytidine and ethenoadenosine dinucleotide compounds of formulas below, wherein LINKER may be attached to the 4- or 5-position of the etheno moiety:
  • LINKER represents any straight chain of 2-24 atoms in length, or aromatic (e.g. phenyl, naphthyl), or heterocyclic rings (e.g. benzothiophene, isoxazole, pyridine, piperidine) optionally substituted and covalently linking the etheno moiety to the FUNCTION moiety;
  • FUNCTION represents any of the methods described herein useful to introduce a heterologous nucleic acid into a cell through intemalization of the receptor-ligand complex;
  • Ri and R 2 may be H, or OH; Additionally preferred compounds are 6 and 8-substituted adenosine triphosphates and their dinucleotides as described in Formula II.
  • Links to the carbons of heterocyclic bases may also be made through photoactivatable linkers, such as, but not limited to the use of the commercially available azidophenyl biotin derivative (Pierce Biochemicals, Immunopure Photoactivatable Biotin).
  • P2 nucleotide ligand conjugates may be formed by attaching a biotinyl moiety through a LINKER or by linking the vector directly through a LINKER to the sugar moiety of the nucleotide, either by derivatization (e.g. acylation, alkylation) of the sugar hydroxyl groups, or through a heteroatom surrogate (e.g. SH, NHR) of the hydroxy groups of molecules represented by formulae I - IV above.
  • derivatization e.g. acylation, alkylation
  • heteroatom surrogate e.g. SH, NHR
  • P2 nucleotide ligand conjugates may be formed by attaching a biotinyl moiety through a LINKER or by linking the vector directly through a LINKER to the terminal phosphate moiety of molecules represented by formulae I, III and IV above.
  • Linking groups used to carry out the present invention are, in general, polymers, including both water soluble polymers and water insoluble polymers.
  • Water soluble, or hydrophilic, linking groups are preferred.
  • the polymers are elongate flexible chains of repeating monomeric units, and may carry or contain functional groups along the chain length thereof.
  • Numerous polymers that can be functionalized to function as linking groups for the ligand and the vector, typically by a covalent bond, are known, and will be readily apparent to those skilled in the art. Examples include, but are not limited to, polysaccharides such as dextran, polyvinyl alcohol, polypeptides such as polylysine, and polyacrylic acid.
  • the ligand and the vector may be bound to the linking group in any conformation or position, including to the free chain end thereof.
  • the linking group will comprise a chain of from 2 to 24 atoms such as carbon atoms, optionally substituted as described above.
  • Biotin can be covalently joined to the ligand by conventional techniques and both biotin groups joined to an avidin or streptavidin group in accordance with known techniques to form a conjugate of the vector and the ligand.
  • ligands to which biotin is covalently joined include:
  • R is H or OH, preferably OH; and n is equal to 1 to 4, preferably 2 or 3.
  • uracil group shown can be replaced with another purine or pyrimidine base as described above, with the biotin and linking polymer chain shown between the biotin group and the uridine group above covalently joined to the purine or pyrimidine base in any suitable position (e.g., a ring carbon such as the 5 carbon in a pyrimidine, or the 2 or 8 carbon in a purine).
  • an oligonucleotide e.g., a DNA, RNA, or chimera of 5 or 10 to 30 or 50 bases
  • an oligonucleotide can be synthesized with one or more bases conjugated to a biotin in this manner, and the thus biotinylated oligonucleotide conjugated to a biotinylated ligand as described herein by means of an avidin.
  • a biotin group can be covalently joined to the vector (particularly vectors having free amine groups such as viral vectors) by means of the EZ-LINK Sulfo-NHS-LC- Biotinylation Kit, available from Pierce (3747 N. Meridian Road, P.O. Box. 117, Rockford, IL 61105).
  • An example of a compound that can be used to biotinylate a primary amine on the vector is Sulfo-NHS-LC-Biotin, available from Pierce, and having the structure:
  • biotin group shown in the sulfo compound described above can be removed and replaced with a covalent linkage to a ligand, as described above, to provide a direct covalent linkage from the vector to the ligand.
  • Hematopoietic stem cells, lymphocytes, vascular endothelial cells, respiratory epithelial cells, keratinocytes, skeletal and cardiac muscle cells, neurons and cancer cells are among proposed targets for therapeutic gene transfer, either ex vivo or in vivo. See, e.g., A.D. Miller, Nature 357, 455-460 (1992); R.C. Mulligan, Science 260, 926-932
  • ⁇ cells and other eukaryotic cells are suitable target cells for the vectors and methods of the present invention.
  • One advantage of the present invention is that it can be used to target heterologous nucleic acids to cells that do not usually bind the transfer vector, i.e, a vims vector.
  • a vims vector any cell that expresses a receptor from the 7-TM receptor family is a suitable target for use according to the present invention.
  • purinoceptors e.g., P2Y ⁇ , P2Y 2 , P2Y , P2Y 6 , P2Yn
  • adenosine receptors i.e., Al, A2, A3
  • bradykinin receptors e.g., BKi , BK ⁇
  • respiratory epithelial cells particularly differentiated columnar airway epithelial cells.
  • the cells may be administered the conjugate in vitro or in vivo, such as by administration of an aerosol containing the conjugate to the luminal surface of airway epithelial cells.
  • purinoceptors that may be used in accordance with the present invention include P2 purinoceptors. Numerous P2 purinoceptors are known. See, e.g., P2 Purinoceptors: Localization, Function and Transduction Mechanisms D. Chadwick and J. Goode Eds 1996.
  • P2 purinoceptors include P2X, P2Y, P2T, P2U (now designated P2Y 2 ) and P2Z purinoceptors (including subclasses thereof). More recently, the purinoceptors have been classified as a P2Y family, consisting of G protein-mediated receptors, and a P2X family, consisting of ligand-gated cation channels. The P2Z purinoceptors, which open non-selective pores, may be considered a third family. Id. at 6-7.
  • the tissue or tissues to be transformed may be determined in part by selection of the purinoceptor to be targeted, and a corresponding ligand selected for the purinoceptor to be targeted, as discussed in greater detail below.
  • P2X ⁇ receptors may be used to target vas deferens (e.g., for production of immunocontraceptive vaccines).
  • Ligands that may be used for this receptor include, but are not limited to, 2-methylthioATP (2-MeSATP), ATP, and ⁇ , ⁇ -methylene ATP ( , ⁇ - meATP).
  • Immune system cells including but not limited to monocytes, macrophages, mast cells, neutrophils and B cells, may be targeted with P2Y and/or P2U receptor ligands, as well as P2Z receptor ligands.
  • Such cells may be transformed with a nucleic acid that expresses an immunogen effective to produce an immune response to an antigen or disease vector in the host subject
  • Pancreatic cells such as pancreatic B cells, may be targeted with P2Y receptor ligands.
  • Such cells may be transformed with a nucleic acid that expresses insulin in the cells in an amount effective to combat hypoglycemia or diabetes in the subject.
  • P2X 3 , P2X ⁇ , P2X 5 , and P2X 6 receptors may be used to target nerve tissue.
  • Ligands that may be used for these receptors include, but are not limited to, 2-MeSATP, ATP, and (for P2X3 and P2X4) ⁇ , ⁇ -meATP.
  • P2Z receptors may be used to target macrophages.
  • Ligands that may be used for these receptors include, but are not limited to, 3'-O-(4-benzoyl)benzoyl ATP (BzATP), ATP, and UTP.
  • P2Y ⁇ receptors may be used to target nerve tissue, placenta and endothelial tissue.
  • Ligands that may be used for this receptor include, but are not limited to, 2-MeSATP, ATP, ADP, and UTP.
  • P2Y 2 receptors may be used to target lung, bone, and pituitary tissue.
  • Ligands include, but are not limited to, ATP, UTP, and 2-MeSATP.
  • P2Y 3 receptors may be used to target nerve tissue.
  • Ligands include, but are not limited to, ADP, UTP, ATP and UDP.
  • P2Y 4 receptors may be used to target placenta and nerve tissue.
  • Ligands include, but are not limited to, UTP, UDP, ATP and ADP.
  • P2Y 5 receptors may be used to target lymphocytes.
  • Ligands include, but are not limited to, ADP, ATP and UTP.
  • P2Y 6 receptors may be used to target blood vessels, particularly smooth muscle and arterial smooth muscle tissue.
  • Ligands include, but are not limited to, UTP, ADP, 2- MeSATP and ATP.
  • P2 purinoceptors include the P2 purinoceptor antagonists, such as: quinidine (P2X and P2Y receptors); imidazolines such as phentolamine (P2Y receptors);
  • PIT 2,2'-pyridylisatogen tosylate
  • P2Y receptors Apamin (P2Y receptors); ⁇ , ⁇ -meATP (P2X receptors); reactive blue 2 (P2Y, P2X and P2Z receptors); suramin (P2X, P2Y, P2T and P2Z receptors); 8- ⁇ ,5-dinitrophenylene carbonylimino)-l,3,5-napthalene trisulfonate, or
  • XAMR0721 P2Y receptors
  • P2X and P2Y receptors pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, or "PPADS”
  • P2X and P2Y receptors pyridoxalphosphate-6-azophenyl-2',5'disulfonic acid, or "isoPPADS”
  • P2X receptors pyridoxal-5-phosphate, or "P5P” (P2X receptors);
  • DIDS 4,4'-diisothiocyanotostilbene-2,2'-disulfonate
  • P2Z receptors Brilliant blue (P2Z receptors); oxidized ATP, or "o-ATP” (P2Z receptors, for targeting macrophages); 2-propylthio-D- ⁇ , ⁇ -difiuoromethylene ATP, or "ARL 66096" (P2T receptors, for targeting platelets).
  • the methods of the present invention provide a means for delivering heterologous nucleic acid independent of the target cell nucleus into a broad phylogenetic range of host cells.
  • the vectors, methods and pharmaceutical formulations of the present invention are additionally useful in a method of administering a protein or peptide to a subject in need of the desired protein or peptide, as a method of treatment or otherwise.
  • the protein or peptide may thus be produced in vivo in the subject.
  • the subject may be in need of the protein or peptide because the subject has a deficiency of the protein or peptide, or because the production of the protein or peptide in the subject may impart some therapeutic effect, as a method of treatment or otherwise, and as explained further below.
  • the gene transfer technology of the present invention has several applications.
  • cDNA or genomic sequences for proteins can be introduced into different cell types in culture, or in vivo, in order to study cell- specific differences in processing and cellular fate.
  • a strong promoter By placing the coding sequences under the control of a strong promoter, a substantial amount of the desired protein can be made.
  • the specific residues involved in protein processing, intracellular sorting, or biological activity can be determined by mutational change in discrete residues of the coding sequences.
  • Gene transfer technology of the present invention can also be applied to provide a means to control expression of a protein and to assess its capacity to modulate cellular events.
  • Some functions of proteins, such as their role in differentiation, may be studied in tissue culture, whereas others will require reintroduction into in vivo systems at different times in development in order to monitor changes in relevant properties.
  • Gene transfer provides a means to study the nucleic acid sequences and cellular factors which regulate expression of specific genes.
  • One approach to such a study would be to fuse the regulatory elements to be studied to reported genes and subsequently assaying the expression of the reporter gene.
  • Gene transfer also possesses substantial potential use in understanding and providing therapy for disease states.
  • diseases There are a number of inherited diseases in which defective genes are known and have been cloned. In some cases, the function of these cloned genes is known.
  • the above disease states fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, at least sometimes involving regulatory or structural proteins, which are inherited in a dominant manner.
  • gene transfer could be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.
  • gene transfer could be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state.
  • the methods of the present invention permit the treatment of genetic diseases, e.g., to screen compounds for activity useful in combatting the disease state.
  • a disease state is treated by partially or wholly remedying the deficiency or imbalance which causes the disease or makes it more severe.
  • the use of site-specific integration of nucleic sequences to cause mutations or to correct defects is also possible.
  • the present invention is employed to express an exogenous CFTR protein in respiratory epithelium.
  • an AdV transfer vector carrying the CFTR gene.
  • the AdV-CFTR is directly linked to the 4-amino substituent of the dinucleotide UP 4 C with a sulfo-NHS activated agent, as described above. Binding of UP 4 C to the P2Y 2 receptor on the apical surface of the respiratory epithelium will induce intemalization of the entire UP 4 C-AdV-CFTR complex into epithelial cells.
  • Suitable subjects to treated according to the present invention include both avian and mammalian subjects, preferably mammalian. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects are preferred. Human subjects of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention. Human subjects afflicted with cystic fibrosis are preferred.
  • the compounds of the invention may be present in the form of their pharmaceutically acceptable salts, such as, but not limited to, an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as manganese, magnesium, or calcium; or an ammonium or tetraalkyl ammonium salt, i.e., NX 4 + (wherein X is C ).
  • Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.
  • Active compounds of the present invention can be administered to a subject in need thereof by any suitable means including oral, rectal, transmucosal, topical or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one may administer the compound in a local rather than systemic manner, for example, in a depot or sustained release formulation. Administration to the lungs is preferred.
  • Active compounds disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by administering an aerosol suspension of respirable particles comprised of the active compound, which the subject inhales.
  • the respirable particles may be liquid or solid.
  • Aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729.
  • Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.
  • Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w.
  • the carrier is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride.
  • Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.
  • Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate medicament aerosol generator.
  • Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration.
  • One illustrative type of solid particulate aerosol generator is an insufflator.
  • Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff.
  • the powder e.g., a metered dose thereof effective to carry out the treatments described herein
  • the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump.
  • the powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant.
  • the active ingredient typically comprises from 0.1 to 100% w/w of the formulation.
  • a second type of illustrative aerosol generator comprises a metered dose inhaler.
  • Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 150 ⁇ l, to produce a fine particle spray containing the active ingredient.
  • Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.
  • the formulation may additionally contain one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.
  • the aerosol may be produced by the aerosol generator at a rate of from about 10 to 150 liters per minute, more preferably from about 30 to 150 liters per minute, and most preferably about 60 liters per minute. Aerosols containing greater amounts of medicament may be administered more rapidly.
  • the dosage of the active compounds disclosed herein or pharmaceutically acceptable salt thereof will vary depending on the condition being treated and the state of the subject, but generally may be an amount sufficient to achieve dissolved concentrations of active compound on the airway surfaces of the subject of from about 10 "9 or 10 "7 to about 10 "3 Moles/liter, and more preferably from about 10 "6 to about 3 x 10 "4 Moles/liter.
  • the daily dose may be divided among one or several unit dose administrations.
  • Other compounds may be administered concurrently with the active compounds, or salts thereof, of the present invention.
  • Solid or liquid particulate pharmaceutical formulations containing active agents of the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 5 microns in size (more particularly, less than about 4.7 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to be deposited in the throat and swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 ⁇ m is preferred to ensure retention in the nasal cavity.
  • the active compounds of the present invention may be administered separately (either concurrently or sequentially) or, alternatively and preferably, they may be pre-mixed and administered as preformed conjugates.
  • a transfer vector carrying a heterologous nucleic acid of interest can be pre-mixed with a targeting molecule (i.e., a bispecific bridging antibody, a peptide, biotin-UTP, etc.) and the complex administered to the subject.
  • a targeting molecule i.e., a bispecific bridging antibody, a peptide, biotin-UTP, etc.
  • active agents or the physiologically acceptable salts or free bases thereof are typically admixed with, inter alia, an acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a capsule, which may contain from 0.5% to 99% by weight of the active compound.
  • One or more active compounds may be incorporated in the formulations of the invention, which formulations may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components.
  • compositions containing respirable dry particles of active compound may be prepared by grinding the active compound with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates.
  • the pharmaceutical composition may optionally contain a dispersant which serves to facilitate the formation of an aerosol.
  • a suitable dispersant is lactose, which may be blended with the active agents in any suitable ratio (e.g., a 1 to 1 ratio by weight).
  • the transfer vectors of the present invention can be used to stably transfect either dividing or non-dividing cells, and stably express a heterologous gene.
  • this vector system it is now possible to introduce into dividing or non-dividing cells, genes which encode proteins that can affect the physiology of the cells.
  • the vectors of the present invention can thus be useful in gene therapy for disease states, or for experimental modification of cell physiology, such as to produce models useful in screening compounds for particular physiologic activity.
  • Epithelial cells are derived from CF and non-CF nasal and bronchial airway epithelia using procedures similar to those described by Gray et al . 1996. Am. J. Respir. Cell Mol. Biol. 14, 104-112. Resected nasal turbinates or portions of mainstem/lobar bronchi representing excess donor tissue are obtained at the time of lung transplantation under the auspices of the University of North Carolina at Chapel Hill Institutional Committee on the Protection of the Rights of Human Subjects. Epithelial cells are removed from the specimens by protease XIV digestion as described (Wu, R., et al., 1985. Am. Rev. Respir. Dis. 132, 311-320), but omitting the filtration step.
  • 1-2 x 10 6 cells are plated per 100mm tissue culture dish in modified LHC9 medium. Lechner, J. F. and Laveck, M. A. 1985. J. Tiss. Cult. Meth. 9, 43-48.
  • the modifications include increasing the EGF concentration to 25ng/ml, adjusting the retinoic acid concentration to 5x 10 "8 M, and supplementation with 0.5 mg/ml bovine semm albumin and 0.8% bovine pituitary extract.
  • the cells are harvested by trypsinization and passage 1 cells are plated at a density of 2.5x10 5 cells on Transwell- Col inserts (Coming-Costar, 24mm diameter, 0.4 ⁇ m pore size), in modified medium.
  • the medium is similar to the supplemented LHC9 except that a 50:50 mixture of LHC Basal (Biofluids) and DMEM-H is used as the base, amphotericin and gentamycin are omitted and the EGF concentration is reduced to 0.5 ng/ml.
  • the apical surface of the cultures are given an air-liquid interface for another 25-30 days until use.
  • Initial histological analyses of human well differentiated (WD) cultures derived from non-CF nasal airways after 34 days of culture indicate that the epithelium is pseudostratified mucociliary, with abundant cilia and cell-types representative of those present in human nasal airways in vivo.
  • AdV adenovims vector
  • Enhancement of AdV-binding above that achieved with a single exposure leads to an increase in gene transfer, since increasing the binding of AdV to cells will increase the probability that an intemalization event leads to AdV entry.
  • PD and WD cultures are exposed to 35 S-Ad5VLacZ (1.2 x 10 10 p) for analyses of AdV- binding, intemalization and transgene expression in the human cultures.
  • 35 S-Ad5VLacZ 1.2 x 10 10 p
  • Radioactive counts per minute (CPM) and ⁇ -gal activity are standardized with respect to the nominal surface area of the culture surface because the apical surface area of cells exposed to vector is the most appropriate denominator, as it allows direct comparison to the epithelium in vivo. It is likely that with PD cells, for a specific incubation time, a 10-fold increase in concentration will result in a 10-fold increase in AdV attachment, intemalization and gene expression as long as saturation of receptor uptake and expression systems does not occur. With WD cultures, although a 10-fold increase in AdV attachment is expected, the corresponding 10-fold increases in intemalization and expression are not. These data indicate that increased binding alone does not overcome the rate-limiting step (intemalization) into WD cultures.
  • CHO and A9 cells both of which are not transducible by AdV
  • HA-tagged human P2Y 2 receptor HA tag on the extracellular N-terminus
  • HA- P2Y 2 receptor expressing A9 cells stain with fluorescently labeled anti-HA Abs under resting conditions.
  • agonist [ATP ⁇ S (10 "4 M)] exposure approximately 80% of the receptors are internalized within 45 minutes. The intemalization of P2Y 2 receptor is mediated via coated pits.
  • Antibody HA.l l(BabCO) against influenza hemagglutin (anti-HA) is directed against the HA-epitope inserted into an extracellular domain of the human P2Y -receptor which is expressed in 1321N1 human astrocytoma cells.
  • the bridging antibody is produced by reacting an anti-fiber (knob) antibody with w-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS, Pierce, Rockford, IL) or N-( ⁇ -maleimidobutyryloxy) sulfosuccinimide ester (Sulfo-GMBS, Pierce, Rockford, IL) at neutral pH. After reduction of anti-HA by mercaptoethylamine, and desalting, the two antibodies are mixed, enabling disulfide cross-link formation. Bifunctional antibody is purified by sequential chromatography over fiber protein and HA columns.
  • the cells were fixed without permeabilization in 4% paraformaldehyde and washed. Monoclonal anti-HA antibody was incubated with the cells followed by incubation with Cy3-conjugated goat anti-mouse IgG secondary antibody (Jackson Immuno Research Labs). The availability of P2Y 2 -HA receptors to the anti-HA was visualized with a fluorescent microscope. The astrocytoma cells were fixed and immunostained for the presence of P2 Y 2 -HA-receptor in the absence of ATP ⁇ S and after 30 min ATP ⁇ S exposure.
  • Confluent MDCK renal cells were used as a model of a polarized, AdV-resistant epithelium.
  • the receptors were localized on the apical surfaces.
  • Cells were exposed to anti-HA and anti-mouse IgG FITC at 4°C, and receptor localization determined by confocal microscopy. It was observed that HA-P2Y 2 receptors in the apical membrane of polarized MDCK cells desensitizes, in part, by intemalization of receptors.
  • Bs-Abs directed to HA- P2Y 2 receptors sequentially administered with AdV-GFP (green fluorescent protein) and ATP ⁇ S transduce HA- P2Y receptor, as compared with Neo-expressing MDCK cells.
  • AdV-GFP green fluorescent protein
  • ATP ⁇ S ATP ⁇ S transduce HA- P2Y receptor
  • A9 cells expressing the HA-epitope tagged BK ⁇ receptor and A9 cells expressing neomycin alone were exposed to bi-specific antibodies (anti-fibre-knob x anti-HA) and AdV in the absence and presence of bradykinin. Briefly, cells at 4°C were incubated in the absence or presence of bi-specific antibody (bs-Ab, lO ⁇ g/ml for 2 hrs), washed and exposed to AdVLacZ (10 10 particles for 2 hrs), washed and exposed to bradykinin (BK, 1 ⁇ M for 2 hrs at 37°C). The cells were then maintained at 37°C for 24 hrs until gene expression was assessed by standard techniques.
  • Figure 5 shows that efficient gene expression occurs only in HAB2k-expessing cells incubated with both bs-Ab and AdV with enhancement of gene transfer by activating the receptor with agonist.
  • CHO cells Choinese Hamster Ovary cells which lack the AdV attachment receptor
  • HA-epitope tagged ⁇ 2 -adrenoreceptor and wild- type (Wt) CHO cells were sequentially exposed to increasing concentrations of bispecific antibodies (anti-fibre-knob x anti-HA) and AdV and finally to isoproterenol.
  • bispecific antibodies anti-fibre-knob x anti-HA
  • AdV wild- type
  • Figure 6A-B shows bs-Ab dose- dependent increases in gene expression only in CHO cells expressing HA-P2Y 2 -R ( Figure 6A) or HA- ⁇ ( Figure 6B) receptors.
  • a prototype agonist-linker is biotin (B)-UTP, which contains a 16 atom linker connecting the 5 position of the pyrimidine base to biotin.
  • B-UTP is an agonist of P2Y 2 receptors ( Figure 7). Fluorescence studies have demonstrated that when CHO cells expressing P2Y 2 receptors are exposed to B-UTP conjugated to streptavidin-Texas Red (TR), the B-UTP triggers and is internalized with the P2Y receptors.
  • HA-P2Y 2 -A9 cells were sequentially exposed to B-UTP, streptavidin (SA), and B-AD (biotin labeled adenovims expressing LacZ). Approximately, 90% of HA-P2Y -A9 cells demonstrated LacZ expression (Figure 8). In contrast, only low levels of LacZ expression were observed in wild-type A9 cells.
  • U 2 P 4 Biologically active analogs of UTP that are more resistant to biological hydrolysis than UTP have been developed for linkage to vectors.
  • the dinucleotide U 2 P 4 has useful properties. As shown in Figure 9, U 2 P 4 is equipotent with UTP in inducing a biological response (inositol phosphate release) and is resistant to hydrolysis in cystic fibrosis (CF) sputum.
  • CF cystic fibrosis
  • Uridine 5'-monophosphate, free acid (1 mmol) (Aldrich, St. Louis, MO) was dissolved in dimethylformamide (DMF, 0.5 mL) and tributylamine (0.5 mL) (Aldrich) and evaporated under reduced pressure to form an oil. The oil was redissolved in dry DMF (0.5 mL) and concentrated again. This evaporation procedure was repeated one more time and the final oil was dissolved in 2.5 mL dry DMF to which was added carbonyldiimidazole (2 mmol) (Aldrich). After stirring at 37°C for 2 hours this stock solution of activated UMP was set aside.
  • DMF dimethylformamide
  • tributylamine 0.5 mL
  • POROS HQ/H column 4.6 x 100 mm, gradient 0-0.66 M ammonium bicarbonate over 20 min, 5.0 mL/min) in ten aliquots.
  • the main fractions from each run (4.7 - 5.4 min) were combined and lyophilized to yield P'-(4-(N'-biotinylaminohexylcarbamoyl- methylthiouridine 5')-P 4 -(uridine 5')tetraphosphate (1.426 mmol, 29.6 % yield, quantitated approximately by comparison of its absorbance at ⁇ ma ⁇ 305 nm with that of a standard solution of 4-methylthiouridine monophosphate).
  • the carboxylic acid from the previous step (6 mg, .00592 mmol) was dissolved in 0.1M 4-morpholinepropanesulfonic acid buffer (1 mL), and LC Biotin Hydrazide (Pierce Chemical, 3 mg, .00807 mmol) added as a solution in dimethylsulfoxide (150 uL). To this was added l-[3-Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (15 uL of a 100 mg/ mL solution in 0.1 M 4-morpholinepropanesulfonic acid buffer; .0078 mmol) and the solution stirred at room temperature for 6 hours.
  • step d is treated with lithium hydroxide/tetrahydrofuran, and coupled to LC Biotin Hydrazide, yielding the corresponding 2'-deoxy cytidine analogue.
  • reaction mixture was extracted with 0.5 N HC1 (75 mL), 50% saturated NaCl (2 x 50 mL), and finally saturated NaCl (25 mL).
  • the organic layer was dried (MgSO4) and was stripped to an oil. After dissolving this residue in toluene (125mL) with heating to 80-90°C, the solution was allowed to stand at room temperature for several days. The resultant crystals were filtered, washed with toluene (5 x 10 mL) and dried at 40°C. The yield was 13.08g (77%).
  • the chloroform layer was dried (MgSO 4 ), stripped and the residue dissolved in chloroform (100 mL), with heating. Hexane was added until the solution just became turbid, and this was allowed to stand overnight at room temperature. The resultant crystals were filtered, washed with 75 chloroform/ 25 hexane (2 x 25 mL) and dried at 40°C. The yield ofpure title compound was 5.6g (45%).
  • P'-(2'-deoxycytidine 5'-)P -(uridine 5'-) tetraphosphate, trisodium salt 100 mg, 0.116 mmol was dissolved in water (1 mL), with stirring .
  • 2-bromo- 4'-(6-bromohexanoyl)amino-acetophenone 210 mg, 0.537 mmol was added as a solution in DMF (2 mL), and the resultant solution was heated overnight at 42°C.
  • the pH was adjusted to 7 by the addition of 1 M sodium bicarbonate, and the reaction mixture partitioned between ethyl acetate (10 mL) and water (15 mL).
  • the aqueous layer was extracted once more with ethyl acetate (10 mL) and was stripped to a foam.
  • the cmde product was dissolved in water (1 mL) and 400 uL of this was purified by sequential injections onto a semi-preparative reverse phase column (Alltech Absorbosphere nucleoside/nucleotide C18, 10x250 mm, 7um; gradient from 0.1 M ammonium acetate to methanol over 30 minutes; 5 mL/minute; monitor at 295nm). The appropriate fractions were pooled, stripped and lyophilized, yielding 18 mg (34%) of the title compound as the tetraammonium salt.
  • the bromide from the previous step (16 mg, 0.0141 mmol) was dissolved in concentrated ammonium hydroxide (2 mL) and stirred overnight at room temperature.
  • the reaction mixture was stripped to a foam, reconstituted in water (500 uL) and the product purified by several sequential injections on the same semi-preparative C18 column used in the previous step, under the same conditions. Stripping and lyophilizing yielded lOmg (66%) of the title compound, as the tetraammonium salt.
  • the amine from the previous step (7 mg, .000654 mmol) was dissolved in water (1 mL) and to this was added succinimidyl-6-(biotinamido) hexanoate (Pierce Chemicals EZ-Link NHS-LC-Biotin, 15 mg, .00329 mmol), as a solution in DMF (2 mL).
  • the pH was adjusted to 8.5 by the addition of 1 M sodium bicarbonate and the mixture stirred for 2 hours at room temperature.
  • the solvents were removed and the product was purified by several sequential injections on HPLC, using the same semi -preparative C18 and conditions as the previo ⁇ s step.
  • the yield of the title compound was 7 mg (76 %), as the tetraammonium salt.
  • the oc ⁇ 3/ 5 integrins are reported to mediate intemalization but not attachment of Ad into epithelial cells in vitro.
  • the localization of the membrane-bound ⁇ y ⁇ 3/ integrins to the apical and/or basolateral membranes of WD cultures is cmcial in understanding the roles of these molecules in AdV-mediated gene transfer.
  • the availability of a number of different antibodies to these integrins allows their location in polarized epithelia to be determined.
  • These integrins are also receptors for peptides containing RGD amino acid sequences.
  • RGD-peptides inhibit AdV-mediated gene transfer to epithelial cells by interaction with the ⁇ 3/5 integrins. This Example illustrates the effects of RGD peptides on AdN-binding, intemalization and transgene expression in PD and WD cultures.
  • proteins were immunoprecipitated and separated by Western analysis on a 4-12% acrylamide gel ( ⁇ ovex) under non-reducing conditions.
  • the biotinylated proteins were probed with streptavidin-conjugated peroxidase secondary antibody and detected by ECL analysis (Supersignal CL-HRP, Pierce).
  • biotinylated proteins identified by R838 are shown in figure 5 (bands at 125kD correspond to the ⁇ y and 97kD are ⁇ 3/5 subunits)and appear to be present in the basolateral membranes of RTE cells and in HeLa cells but absent from the apical membrane of the WD cultures, suggesting that rat vitronectin receptors may not be located apically in these culture-types.
  • the absence of these integrins in the apical membrane could account for the low rate of AdN-internalization into these cultures.
  • Proteins from the apical and/or basolateral membranes of human PD and WD cultures are selectively isolated by exposing individual surfaces to Sulpho- ⁇ HS-Biotin at 4°C.
  • Standard immunoprecipitations experiments are performed with selective human antibodies (LM609, ⁇ v ⁇ 3 ; P1F6, ⁇ v ⁇ 5 ; V ⁇ R147, ⁇ v ; P4Gl l, ⁇ ; CD61, ⁇ 3 ; anti- ⁇ 5 and R838, ⁇ 3 ; all obtained from Chemicon Inc, CA.). This procedure allows for detection and localization of the oty ⁇ integrins to the apical and/or basolateral membrane.
  • Adenoviral attachment, intemalization and transgene expression in PD and WD cultures is measured, as described above, in the absence and presence of RGD-peptides.
  • Hexa-peptides, the bioactive GRGDSP (Gibco-BRL) and the inactive control peptide GRGESP (Gibco-BRL) are administered to PD and WD cultures at a final concentration of 0.1-4.0 mg/ml for 2 hrs at 4°C before the addition of AdV (10 10 p/ml).
  • Cyclical RGD peptides (Immunodynamics, La Jolla, CA.), reported to be more potent at reducing AdV- mediated gene transfer, are also used. Analyses of AdV-attachment, intemalization and transgene expression are performed as described above.
  • knob protein is conjugated to fluorescent microspheres of the same diameter as Ad and the initial interactions of the knob-spheres on PD and WD cells are assessed by confocal microscopy. Increased specific or non-specific binding may increase intemalization and possibly expression.
  • AdV Intemalization into HeLa Cell-Lines with Competent and Defective Receptor-Mediated Endocytosis Adenoviral entry into cells may reflect uptake by a number of cellular pathways i.e., receptor-mediated endocytosis via coated pits, non-specific pinocytosis, or phagocytosis.
  • receptor-mediated endocytosis via coated pits, non-specific pinocytosis, or phagocytosis.
  • HeLa cell mutants have been produced which can overexpress either wild-type dynamin protein or a mutant form, mDyn (controlled by a TET-inducible promoter).
  • dynamin is responsible for coated pit endosome formation and functions by 'pinching' off invaginations in the plasma membrane.
  • HeLa cells overexpressing wild-type dynamin show no functional or morphological alteration of uptake processes compared to parent cells.
  • Cells overexpressing mDyn form coated pits and invaginate the plasma membrane but fail to bud coated vesicles into the cytoplasm.
  • Ligands for receptor-mediated endocytosis (EGF and transferrin) fail to be internalized into cells expressing mDyn, but ligand-receptor binding, coated pit assembly, recmitment of receptors into coated pits and invagination of the plasma membrane are all unaffected. In the absence of receptor-mediated endocytosis, non-specific pinocytosis initially remains unaltered but with time is upregulated to compensate for the loss of receptor-mediated endocytosis.
  • EXAMPLE 18 Analysis of Transgene Expression in Human PD and WD Cultures PD and WD cultures are exposed to Ad5VLacZ by application to either the apical and/or basolateral membranes over a range of viral titres (10 6 -10 n infectious units/ml: corresponding to MOI range of 1-10 5 ) with incremental exposure times (1-24 hrs), to study the effects of concentration and time on the gene expression obtained.
  • Vectors used in this study are produced and titred by the UNC Gene Therapy Core. Incubations are performed at 37°C and/or 4°C.
  • the former temperature allows potential cellular uptake processes to be studied, while the latter temperature, is a standardized technique for measuring the initial attachment of ligands to their receptors, in the absence of receptor recycling and/or intemalization.
  • Gene expression is assessed 48 hrs after initial exposure to AdV by both qualitative and quantitative means (X-gal histochemistry and standard colourimetric enzyme assays, respectively). See Pickles, R. J., et al., 1996. Human Gene Therapy 7, 921 -931.

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Abstract

La présente invention concerne une méthode d'apport à une cellule d'un acide nucléique hétérologue (par exemple, une séquence génique), consistant à fixer un virus renfermant une séquence génique hétérologue sur un récepteur couplé à une protéine G (à savoir, un récepteur à sept membranes, tel que le récepteur P2Y2). Le virus peut être fixé sur un récepteur par un anticorps de formation de ponts, ou par la liaison d'un anticorps spécifique du récepteur à un anticorps spécifique du virus, l'anticorps se liant spécifiquement au récepteur et l'anticorps se liant spécifiquement au virus étant tous deux réticulés. Selon un autre mode de réalisation, le virus peut éventuellement exprimer un peptide se liant spécifiquement au récepteur. Le récepteur peut être amené à s'internaliser par l'addition d'un ligand connu pour déclencher l'internalisation du récepteur dans la cellule.
PCT/US1999/030658 1998-12-23 1999-12-22 Transfert genique cible utilisant des recepteurs couples a une proteine g WO2000039145A1 (fr)

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US6319908B1 (en) 1996-07-03 2001-11-20 Inspire Pharmaceuticals, Inc. Method for large-scale production of di(uridine 5′-tetraphosphate) and salts thereof
US6548658B2 (en) * 1997-07-25 2003-04-15 Inspire Pharmaceuticals, Inc. Di-(uridine 5′)-tetraphosphate and salts thereof
US6617444B1 (en) * 1999-06-30 2003-09-09 Yamasa Corporation Dinucleotide tetraphosphate crystals
US6703376B2 (en) 1996-07-23 2004-03-09 Inspire Pharmaceuticals, Inc. Use of uridine triphosphates and related compounds for the prevention and treatment of pneumonia in immobilized patients
US6713458B1 (en) 1997-07-25 2004-03-30 Inspire Pharmaceuticals, Inc. Therapeutic uses of di(uridine 5′)-tetraphosphate and salts thereof
US6872710B2 (en) 1997-07-25 2005-03-29 Inspire Pharmaceuticals, Inc. Di(uridine 5′)-tetraphosphate and salts thereof
US7018985B1 (en) 2000-08-21 2006-03-28 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7132408B2 (en) 2000-08-21 2006-11-07 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7223744B2 (en) 1997-02-10 2007-05-29 Inspire Pharmaceuticals, Inc. Pharmaceutical formulation comprising dinucleoside polyphosphates and salts thereof
US7432252B1 (en) 1997-07-25 2008-10-07 Inspire Pharmaceuticals, Inc. Method of promoting cervical and vaginal secretions
US7452870B2 (en) 2000-08-21 2008-11-18 Inspire Pharmaceuticals, Inc. Drug-eluting stents coated with P2Y12 receptor antagonist compound

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US5789391A (en) * 1996-07-03 1998-08-04 Inspire Pharmaceuticals, Inc. Method of treating sinusitis with uridine triphosphates and related compounds
WO1998034942A2 (fr) * 1997-02-06 1998-08-13 Inspire Pharmaceuticals, Inc. Dinucleotides et leur utilisation en tant que modulateurs d'epuration mucociliaire et de frequence des battements ciliaires
US5837861A (en) * 1997-02-10 1998-11-17 Inspire Pharmaceuticals, Inc. Dinucleotides and their use as modulators of mucociliary clearance and ciliary beat frequency
US5900407A (en) * 1997-02-06 1999-05-04 Inspire Pharmaceuticals, Inc. Method of treating dry eye disease with uridine triphosphates and related compounds
US6022527A (en) * 1997-08-29 2000-02-08 The University Of North Carolina At Chapel Hill Method of treating lung diseases with uridine 5'-diphosphate and analogs thereof

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US5789391A (en) * 1996-07-03 1998-08-04 Inspire Pharmaceuticals, Inc. Method of treating sinusitis with uridine triphosphates and related compounds
US5958897A (en) * 1996-07-03 1999-09-28 Inspire Pharmaceuticals, Inc. Method of treating sinusitis with uridine triphosphates and related compounds
US5972904A (en) * 1996-07-03 1999-10-26 Inspire Pharmaceuticals, Inc. Method of treating sinusitis with uridine triphosphates and related compounds
WO1998034942A2 (fr) * 1997-02-06 1998-08-13 Inspire Pharmaceuticals, Inc. Dinucleotides et leur utilisation en tant que modulateurs d'epuration mucociliaire et de frequence des battements ciliaires
US5900407A (en) * 1997-02-06 1999-05-04 Inspire Pharmaceuticals, Inc. Method of treating dry eye disease with uridine triphosphates and related compounds
US5837861A (en) * 1997-02-10 1998-11-17 Inspire Pharmaceuticals, Inc. Dinucleotides and their use as modulators of mucociliary clearance and ciliary beat frequency
US6022527A (en) * 1997-08-29 2000-02-08 The University Of North Carolina At Chapel Hill Method of treating lung diseases with uridine 5'-diphosphate and analogs thereof

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6319908B1 (en) 1996-07-03 2001-11-20 Inspire Pharmaceuticals, Inc. Method for large-scale production of di(uridine 5′-tetraphosphate) and salts thereof
US6703376B2 (en) 1996-07-23 2004-03-09 Inspire Pharmaceuticals, Inc. Use of uridine triphosphates and related compounds for the prevention and treatment of pneumonia in immobilized patients
US7223744B2 (en) 1997-02-10 2007-05-29 Inspire Pharmaceuticals, Inc. Pharmaceutical formulation comprising dinucleoside polyphosphates and salts thereof
US7091334B2 (en) 1997-07-25 2006-08-15 Inspire Pharmaceuticals, Inc. Method for large-scale production of di(uridine 5′)-tetraphosphate and salts thereof
US7528119B2 (en) 1997-07-25 2009-05-05 Inspire Pharmaceuticals, Inc. Di(uridine 5′-)tetraphosphate and salts thereof
US6765090B2 (en) 1997-07-25 2004-07-20 Inspire Pharmaceuticals, Inc. Method for large-scale production of di(uridine 5')-tetraphosphate and salts thereof
US6872710B2 (en) 1997-07-25 2005-03-29 Inspire Pharmaceuticals, Inc. Di(uridine 5′)-tetraphosphate and salts thereof
US6713458B1 (en) 1997-07-25 2004-03-30 Inspire Pharmaceuticals, Inc. Therapeutic uses of di(uridine 5′)-tetraphosphate and salts thereof
US7939510B2 (en) 1997-07-25 2011-05-10 Inspire Pharmaceuticals, Inc. Di(uridine 5′-)tetraphosphate and salts thereof
US7132410B2 (en) 1997-07-25 2006-11-07 Inspire Pharmaceuticals, Inc. Di(uridine 5′-)tetraphosphate and salts thereof
US6548658B2 (en) * 1997-07-25 2003-04-15 Inspire Pharmaceuticals, Inc. Di-(uridine 5′)-tetraphosphate and salts thereof
US7432252B1 (en) 1997-07-25 2008-10-07 Inspire Pharmaceuticals, Inc. Method of promoting cervical and vaginal secretions
US6617444B1 (en) * 1999-06-30 2003-09-09 Yamasa Corporation Dinucleotide tetraphosphate crystals
US7018985B1 (en) 2000-08-21 2006-03-28 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7452870B2 (en) 2000-08-21 2008-11-18 Inspire Pharmaceuticals, Inc. Drug-eluting stents coated with P2Y12 receptor antagonist compound
US7618949B2 (en) 2000-08-21 2009-11-17 Inspire Pharmaceuticals, Inc. Drug-eluting stents coated with P2Y12 receptor antagonist compound
US7132408B2 (en) 2000-08-21 2006-11-07 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation

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