CA2295315A1 - Targeted gene transfer using g protein coupled receptors - Google Patents

Abstract

A method of delivering heterologous nucleic acid (e.g., a gene sequence) into a cell comprises attaching a virus containing a heterologous gene sequence to a G protein coupled receptor (i.e., a seven transmembrane receptor such as the P2Y2 receptor). The virus may be attached to the receptor by means of a bridging antibody, or by binding an antibody specific for the receptor with an antibody specific for the virus, wherein the antibody that specifically binds with the receptor and the antibody that specifically binds to the virus are cross-linked. Alternatively, the virus may express a peptide that specifically binds to the receptor. The receptor may be induced to internalize by means of the addition of a ligand known to trigger internalization of the receptor into the cell.

Description

TARGETED GENE TRANSFER USING G PROTEIN
COUPLED RECEPTORS
RELATED APPLICATION INFORMATION
This application claims the bene it of United States Provisional Application No. 60/050,843 filed June 26, 1997.
STATEMENT OF FEDERAL SUPPORT
This invention was made with Government support under Grant No.
HL51818 from the National Institute of Health. The government has certain rights in this invention.
~o FlELD OF THE INVENTION
This invention relates to methods and systems useful in the transfer of nucleic acids into eukaryotic ceils.
BACKGROUND OF THE INVENTION
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.
2o The introduction of a particular foreign or native gene into a mammalian host cells is facilitated first by introducing a gene sequence

2 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. In contrast to methods which involve DNA transformation or transfection, the use of viral vectors can result in the rapid introduction of the recombinant molecule in a wide variety of host cells. In particular, 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 1o 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: Acad. Sci. USA 84, 4626 {9987)), adeno-associated virus (AAV; see, e.g., N. Muzyczka et al., J. Clin. invest. 94, 1351 (1994)); herpes simplex virus (see, e.g., A.I. Geller, et al., Science 241, 1667 (1988)), and others.
Efforts to introduce recombinant molecules into mammalian cells have been hampered by the inability of many cells to be infected by the above-described viral or retroviral vectors. Limitations on retroviral 2o vectors, for example, 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).
Accordingly, there exists a need in the art for improved methods of introducing and expressing genes in target cells.
SUMMARY OF THE INVENTION
The shortcomings of current methods of receptor-mediated gene transfer are overcome by the methods and complexes of the present 3o invention. In particular, 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 internalization 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 internalization 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.
BRIEF DESCRIPTION OF THE DRAWINGS
o 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 ~5 the present invention.
Figure 3 is a graphical representation of the Cf secretory responses of human airway epithelia to lumenal NECA (A2b agonist), isoproterenol, bradykinin, or ATP (all 10'~ M).
Figure 4A is a graphical representation of the dose-effect relationship between bs-Ab concentration and gene transfer efficiency in A9-null cells ( ~ ) compared with HA-P2Y2-A9 administered sequentially (O) or as preformed conjugates (1).
Figure 4B is a graphical representation of a study to evaluate the specificity of increased gene transfer with bs-Ab in A9-HA-P2Y2 and A9-null cells pre-treated with specific or non-specific bs-Ab or after chronic desensitization of HA-P2Y2 receptors by pretreatment with ATPyS.
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 P2Y2 and ~i2 receptors and adenovirus fiber protein.
Figure 7 is a graphical representation of biotin-UTP stimulation of inositol phosphate formation in P2Y2 receptor expressing ( ~ ) but not wild-type (~) astrocytoma cells.
Figure 8 is a graphical representation of the stimulation of gene o transfer in A9 (wt) and HA-P2Y2-A9 cells in the presence of biotin-UTP
conjugated by streptavidin to biotin-Ad.
Figure 9A is a graphical representation of a comparison of agonist potency of U2P4 and UTP in astrocytoma cells expressing P2Y2 receptors.
Figure 9B is a graphical representation of the metabolic stability of U2P4 compared with UTP in cystic fibrosis sputum.
DETAILED DESCRIPTION OF THE INVENTION
2o The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout.
A transfer vector-receptor complex of the present invention comprises a transfer vector bound to a receptor that is capable of being 3o 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. In particular preferred embodiments, described in more detail hereinbelow, the transfer vector is targeted to a seven WO 99/00511 PC'T/US98/13336 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 s vector, a plasmid, an oligonucleotide, or RNAIDNA chimeric molecules, as is described more fully hereinbelow. Interaction between the 7-TM
receptor and the targeted complex results in receptor complex internalization, thereby introducing the heterologous nucleic acid carried by the transfer vector into the cell where it is expressed.
Viral Transfer Vectors of the Present Invention One embodiment of the invention is described with reference to Figure 1. In this embodiment, a complex (i.e., a conjugate) of the present invention comprises a viral vector 10 (which is illustrated in the figure as 1s an adenovirus) 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.
Although the viral vector 10 is illustrated as an adenovirus 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 retrovirus (i.e., Maloney virus such as Moloney Murine 2s Leukemia Virus and lentiviruses) vectors, adeno-associated virus (AAV) vectors, and herpes virus vectors (Figure 2). The viral vectors of the present invention may be attenuated viruses or may be rendered non-replicative by any method known to one skilled in the art.
However, the use of adenoviruses as the vector is currently so preferred.
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 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 adenovirus genome). The term "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 o sequences may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such 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.
Standard techniques for the construction of the vectors of the present invention are well-known to those of ordinary skill in the art and 2o can be found in such references as Sambrook et al., Molecular Cloning: A
Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989). A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and which choices can be readily made by the skilled artisan.
As will be appreciated by one skilled in the art, the nucleotide sequence of the inserted heterologous gene sequence or sequences may be of any nucleotide sequence. For example, the inserted heterologous gene sequence may be a reporter gene sequence or a selectable marker gene sequence. A reporter gene sequence, as used herein, is any gene 3o 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. Alternatively, 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. Examples of 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 0 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 IacZ gene which encodes the beta-galactosidase activity of E. coli; and the gene encoding puromycin resistance.
~ 5 Preferred reporter or selectable marker gene sequences are sufficient to permit the recognition or selection of the vector in normal cells.
In one embodiment of the invention, the reporter gene sequence will encode an enzyme or other protein which is normally absent from mammalian cells, and whose presence can, therefore, definitively 2o 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. Preferably, the 25 heterologous gene sequence encodes a therapeutically active protein or poiypeptide. In one particular preferred embodiment, the heterologous gene sequence encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein or biologically active analogs, fragments, or derivatives thereof. Alternatively, the heterologous gene sequence may 3o 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 poiynucieotide in the cells of the individual.

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f.~cprenlon of the heteroloaous dens may prov~do Immunogenic dr antlpANa protein or polypopptids to schleve an an#body rwponao, which antlbodlee can be odiacted fioM an anirnsl In a body fluid such as blood, serum or a1oltss.
It ts, of ovune, poe~Ibla to employ, se the Insw~sd heteroiopoua gene sequenos, a glno sequence which already pos~rsses~a promoter, Initiation sequence, or processing ssquenvo.
Non-YI~ Yeators at the Present enwhtlon In olhsr prsi~eirsd embod~rrwnts, tho transfer vector Is nonwiral, Other s~rtable vlcton InGude, but aro not limited to, ollponudeotidss (Indudlng RNA. DNA, synthetic end mod~flad nuolela adds), plasmlds. Fund RNAIDNA chlmvrin moiecuiee as de~tGrl~d by ~. Komotx, oligonudv~de vdcbors Itlduds an4sensp oliponudoo#dvo and otlponuoieot<dow that ~lrns~lon as nbotyrrats. The non-vlrill ttensEer wotors of the proaent irNerdton are able to -induda ~snous r~ude~c actdv os described herolnabvw with roepe4t bo virtl l~sorooro, Oilaonuoieotldes, plaarnids, end RN~tDNA chimsdo moloa~ ott~ be synthesized or produovd by any sulhbla m~had knawn In the art.
Hewn Trnnsm~rtblene R~loaptoea of !hs P~ss4n! imrenUon Receptors wcoordinp tO the prlant tnven#on betanp bo the ferr~ily of T-TM receptors. See psn~J~y, 8. Vltatson et al,, The Q-Pro~rlrr Linked Receptor FsctaBook, Aaadvmlc Press, Now York (1894); U.B. Patertt No.
6,482,8# to Klng et at, Those skHled in the srt vet a~pprrctats that T-TM reCSptotl ere O
protein c4upled reo~ptoro. Any mammailah G protafi onuplvd rooeptor, and the nude'ro tad sequences encoding t>leae receptors, may bo employed ~in prncdalng the present Inven~on. Examplso of each rserptars inctude, but arcs not llrnitsd tp, dopamine receptors, musoarinia ahollne~lc reaepoon3. Q-adrenerpfc rscopbate, opistl r~saep~or~t, t~nnablrrold r~eosptora, ser~ooonin rooepbore, p.a~drener~gta reoeptcrs, dnd purindcepton.
'fhe term "reaepeo~' ss used haroirl,l~ fn~ended 1A enoompsss subtypes of - w~
s ,~ ~ g x ~nroc ~ a ~r~. ~~, ...~, ~~t 'd a56r, '~~

the named receptors, and mutants and homologs thereof, along with the nucleic acid sequences encoding the same.
Preferably, the 7-TM receptor for use according to the present invention is a purinoceptor (e.g., P2Y~, P2Y2, P2Y4, P2Ys and P2Y~~), an adenosine receptor (i.e., A1, A2, and A3, and sub-types thereof), a bradykinin receptor (e.g., BK, and BK"), or a ~i-adrenergic receptor (e.g., ~3~, X32 and ~i3). Also preferred is the C5A complement receptor. More preferred are the P2Y2, BK,i, A2B, X32, and C5A receptors, with the P2Y2 receptor being most preferred. Thus, ligands that may be used to carry 0 out the present invention include nucleotides, nucleosides, catecholamines (e.g., dopamine, 5-hydroxytryptophan), CSA, and bradykinin(s).
The P2Y2 (also known as the P2~)- purinoceptor undergoes internalization 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. PharmacoG
103, 1649-1656. Molecular conjugation of AdV to P2Y2-receptors, followed by activation of these receptors by ATP/UTP, leads to internalization of the vector-ligand-receptor complex into endosomes and thus provide an alternative entry pathway for AdV into the WD epithelium, and thereafter to 2o gene expression.
Antibodies of the Present Invention As shown in Figure 1 and Figure 2, one strategy for targeting transfer vectors carrying heterologous nucleic acids to 7-TM receptors for internalization into the cell is with a bispecific bridging antibody. In general, 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 internalization of the receptor. The bound antibody-transfer vector complex is internalized along with the 7-TM receptor, thereby introducing the transfer vector carrying the heterologous nucleic acid into the cell. According to this embodiment of the invention, the transfer vector is preferably a viral vector, more preferably, AdV, and the bispecific antibody comprises a monoclonal antibody directed against the fiber (knob) protein of the s adenovirus.
The term "antibodies" as used herein 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) ~ o 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 Segel et al., U.S. Patent No. 4,676,980.
Antibodies may be polyclonal or monoclonal, with monoclonal being preferred. In particular embodiments, the antibodies are bridging antibodies that are specific to both the target receptor and the transfer 2o vector. According to this embodiment, the bridging antibody is preferably a monoclonal antibody directed to the adenovirus fiber (knob) protein.
Also preferred are monoclonal antibodies and bridging antibodies comprising monoclonal antibodies that are directed to specific epitopes of the 7-TM receptor of interest.
2s Antibodies that bind to the epitope (i.e., the specific binding site) that is bound by the 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 3o 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 s be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265, 495-97 (1975). For example, 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 o 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 s skilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989).
Antibodies specific to the 7-TM (e.g., P2Y2 ) receptor: may also be obtained by phage display techniques known in the art.
Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for 2o carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980)(CRC Press, Inc., Boca Raton, FL); see also U.S. Patent No. 4,727,022 to Skold et al. titled "Methods for Modulating Ligand-Receptor Interactions and their Application," U.S.
Patent No. 4,659,678 to Forrest et al. titled "Immunoassay of Antigens,"
2s U.S. Patent No. 4,376,110 to David et al., titled "Immunometric Assays Using Monoclonal Antibodies," U.S. Patent No. 4,275,149 to Litman et al., titled "Macromolecular Environment Control in Specific Receptor Assays,"
U.S. Patent No. 4,233,402 to Maggio et al., titled "Reagents and Method Employing Channeling," and U.S. Patent No. 4,230,767 to Boguslaski et 3o al., titled "Heterogenous Specific Binding Assay Employing a Coenzyme as Label." Applicants specifically intend that the disclosures of all U.S.
Patent references cited herein be incorporated herein by reference.
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., 355 ~2s1, ~3~1), enzyme labels (e.g., horseradish peroxidase, alkaline s phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques. The term "antigenic equivalents" as used herein, 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.
Non-Antibody Based Targeting Strategies The invention has been described above with respect to bispecific bridging antibodies as means of targeting the transfer vector to the 7-TM
~ s receptor for internalization. As shown in Figure 2, alternative targeting strategies include those utilizing peptides and 7-TM receptor agonistlantagonists.
With respect to peptides, the peptide can be a natural ligand that binds to the 7-TM receptor. Peptide agonists and antagonists of 7-TM
2o receptors are known in the art. Additionally, novel 7-TM receptor agonists/antagonists can be identified as described by U.S. Patent No.
5,482,835 to King et al. Alternatively, the peptide can be identified by phage display techniques, or any other method in the art, as binding to the 7-TM receptor.
2s Methods of synthesizing or producing peptides are welt-known in the art. In one particular embodiment, 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 30 or the H1 loop region of the knob protein. In alternate embodiments, concatomers of the peptide are expressed in the knob protein.
As a further alternative, targeting can be achieved with peptides incorporated into "receptorbodies". In general, a receptorbody is a truncated receptor in which a peptide that binds to a 7-TM receptor is substituted for the intracellular region of the receptor. For example, a truncated BK" receptor can be fused to a peptide ligand for a 7-TM
receptor. This complex will bind to a recombinant AdV expressing the bradykinin peptide in the knob protein region by way of the truncated s receptor. The peptide ligand fused to the truncated BK" receptor will then target this complex to its cognate receptor on the cell surface.
As yet a further alternative, the transfer vector can be targeted to the 7-TM receptor by a chemically-linked high affinity agonistlantagonist of the 7-TM receptor. High-affinity agonists/antagonists may be peptide o ligands, as described above. Alternatively, they are other molecules, such as ATP, UTP, dinucleotides (described in more detail hereinbelow), and derivatives thereof.
In one particular embodiment, a biotin (B) -UTP is used as a targeting agonist for the P2Y2 receptor. The B-UTP can interact with a s biotinylated viral transfer vector in the presence of streptavidin {SA) to give a virus-biotin-SA-biotin-UTP complex that will be targeted to the P2Y2 receptor. Alternatively, oligonucleotides, plasmids, and RNA/DNA
chimeric molecules can be synthesized or produced to incorporate B-UTP
or any other suitable labeled nucleotide.
2o In a further alternative embodiment, high affinity agonists or antagonists can be directly linked to the transfer vector using sulfo-N-hydroxysuccinimide (NHS) as described in more detail below.

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' ,.. --, ~! ~ ~ ,~ a N X999 divtln and Covehnt Cor~u~sbr Fxomptl~ of compotJnda that can bs used to carry out the pnaent invention include thosa havlnd the qenent tonnuls; ,.
. .
A -O H
n wheroin:
A is a pu~ine or pyrlmidlne boas te.a., adenine, guanine, thymine, cyDoslns, undt)~sach patina or pytimldlna bass is proferabiy ~otr~ed to the ribose or deoxyriboaa tins by cavalant bend bo the 6 nftrogetr in the oase of purinle, of by oovsietlt bond to the 1 nltcogen fn the ce~e of pyrtrrddlnea):
R, le H or OH; ~rtsct n la from t to 8, pnrferebly 9 or 4. The trsna~r veceor to oovalently or nonoovalandy )olned or cor~upAted bo the patina or pyrtmidlne base, or the corroapondlng ribdre or deoxyriboea rir~~, by any suitable means, ouch at by aov~tienHy jolr~lng a llnldnp potynror chain therrbo in dny sultobio position (e,p.. a rlr>g c11'bon ouch as the 5 carbon (n a pyrlmidtne, or the ~ or 8 oacbott in r purlne~, bo whloh ilnldnd group the llgend mery be oovtter~t~r allac~ed, ortD which tinidr~ group w blaNn prplfp may be athched, with a bbtin group oo~tarrby ~otr»d eo iha lig$nd (aes below) and tha twp blotM groups jobled to ona another by means of an avidtn proWp to whtct~ both biotin groups ana joined or conjugated.
9ped~c examples Of lipsnds that ~n bo ufed to carry out tha prr4int lrwer~tion lnotude thoaa having the general formula;
whsretn~.

~. ,..-...-.i,v ~a..,~99st ~a~L... . .. s ~ s s R >iareE~s 66st ~t:a ~N~r A and B are each independently a purine or pyrimidine base (e.g., adenine, guanine, thymine, cytosine, uracil); preferably, A is uracil and B is cytosine;
R~ and R2 are each independently selected from the group 5 consisting of H or OH;
n is from 1 to 6, preferably 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 o manner as described above.
Linking groups used to cant' out the present invention are, in general, polymers, including both water soluble polymers and water insoluble. Water soluble, or hydrophilic, linking groups are preferred. The polymers are elongate flexible chains of repeating monomeric units, and s 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 2o 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. In general, the linking group wilt comprise a chain of from 2 to 24 carbons, optionally substituted as described above.
2s Biotin can be covalently joined to the ligand by conventional techniques and both biotin groups joined to an avidin or strep-avidin group in accordance with known techniques to form a conjugate of the vector and the ligand.

Examples of ligands to which biotin is covalently joined include:

HN~NH 0 H H
NH
SI O
wherein R2 is H or OH, preferably OH; and n is equal to 1 to 4, preferably

3. Such compounds are known and commercially available. The uridine 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 ~ 5 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).
Significantly, an oligonucleotide (e.g., a DNA, RNA, or chimera of 5 or 10 to 30 or 50 bases) can be synthesized with one or more bases conjugated to a biotin in this manner, and the thus biotinylated oligonucleotide 2o 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-LINKT"" Sulfo-NHS-LC-Biotinylation Kit, available from Pierce (3747 N.
25 Meridian Road, P.O. Box. 117, Rockford, IL fi1105). 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:

O ~' _NH
30 Na03S
O O H H
II II
O-C-(CHz)5-NH-C-(CHZ)4 S
O

In an alternate embodiment, the 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.
Target Cells of the Present Invention Hematopoietic stem cells, lymphocytes, vascular endothelial cells, respiratory epithelial cells, keratinocytes, skeletal and muscle cardiac cells, neurons and cancer cells are among proposed targets for therapeutic o 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 (1993). These 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 s not usually bind the transfer vector, i.e, a virus vector.
In particular, any cell that expresses a receptor from the 7-TM
receptor family is a suitable target for use according to the present invention. Preferred are cells that express purinoceptors (e.g., P2Y~, P2Y2, P2Y4, P2Ys, P2Y»), adenosine receptors (i.e., A1, A2, A3), 2o bradykinin receptors (e.g., BK, , BK"), or (i-adrenergic receptors (e.g., (i~, ~2, ~3). More preferred are cells that express P2Y2, BK", A2B, ~Z, Csa, receptors, with cells that express the P2Y2 receptor being most preferred.
Also preferred as targets are respiratory epithelial cells, particularly differentiated columnar airway epithelial cells. The cells may be 25 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.
Gene Transfer Technology 3o 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. in this manner, the protein or peptide may thus be produced in vivo in the subject. The subject may be in need of the protein or peptide s 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. The most immediate applications are perhaps in elucidating the processing of peptides and functional domains of proteins. Cloned 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. By placing the coding sequences under the s control of a strong promoter, a substantial amount of the desired protein can be made. Furthermore, 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 2o 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 2s 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 3o expression of the reporter gene.
Gene transfer also possesses substantial potential use in understanding and providing therapy for disease states. 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. In general, 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.
s For deficiency state diseases, 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. For unbalanced disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to o counteract the disease state. Thus the methods of the present invention permit the treatment of genetic diseases. As used herein, 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 ~ 5 defects is also possible.
In one particularly preferred embodiment, the present invention is employed to express an exogenous CFTR protein in respiratory epithelium. According to this embodiment, it is preferred to use an AdV
transfer vector canying the CFTR gene. The AdV-CFTR is directly linked 2o to the dinucleotide UP4C by a sulfo-NHS linker, as described above.
Binding of UP4C to the P2Y2 receptor on the apical surface of the respiratory epithelium will induce internalization of the entire UP4C-sulfo-NHS-AdV-CFTR complex into epithelial cells.
2s Pharmaceutical Formulations, Subjects, and Methods of Administration 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 3o 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.

Active compounds of the present invention may be prepared as pharmaceutically acceptable salts thereof. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of s such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, malefic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, ~o benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine.
Active compounds of the present invention can be administered to ~5 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 2o 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.
so 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 SENT BY:SIMBAS . _ _ ~ 1-~5 uu ~2~9«~~s 1999-i2-is SIMBAS-~ 819 953 95~
. . . ~i!!~ 9 ~ J 13 ,~ ~ ~''~A~B , v ! U ~ t999 M a flqu(d canter, the rdhrs InQnedient oomprtslng up to d0~ wlw of the tontntadon, but pndvrsbty leas than 20% wlw. 'frie carrlor to typlcaily water (and moat prefrnbly :~rtle, pyropenfrgs avatar) ar a dil~rte e~qwous a~cahoiic ~otutlon, pnierebly mivda (eatontc with body ~ulds by the e;ddftfon oi, ibr example, wdlum chloride, Optiorwl addl'ttvvss Include prrservatlves if the i~ormuiabon Is not rnwda a~tarlie, for example, mettrr! hydroxybstuo~, arrtlaxldants, flavoring agenb, volat0e oils, 6utferlng agents and surfe~ctants.
Aen~ots of solid psrbdee oomprlsihQ tha active compound may likewise be produced w~h any solid particulate medicament aerosol penenbor. Aerosol peneratoro fur rdmlnlatsrlng solkl pertloulate rnedioamsnb to a eubjeot produce pat~des nrhtoh are n~pirabN, ae explained above, and 9eneraEs a v4lums of aerosol oonteintnp a prodetirMined metered doae of a ttbdbprnon! st s raita sultsble for human e~dminislntion. pre IUusttatlva type of eoikf paWculabr aerosol penorator is an lnsulAshor. 8ultsble lbrtnuiwtione for admlnfstratfon by lneufrtatlon indude ilnsly commlnuhd powder8 which fist' be delivered by msarn o~ an lnsufrie~r or taksa Into the nasal aavlty in the manner of a Bnuif, !n the Irreuftlebr, the powder (a.g" a metered down thereof ethcttvs to carry out the treatments described her~)'is cvrtatnsd In ~poulea or cerMdpos, typiaatly mode of pslaln or plse~c, which sfie eftllar plarasd or opendd In Htu and the powdeN 4eNwrod by air drawn thro~t~h the device upon ihhalatbn or by moans of t manuatly.oper~ated pump, 'rha powder err~pioysd tn the ineufiatipr consists either svlvly df the actiVS Ingrvdfsnt or of a powder blend cornpttslr~ the ectlvl ir~rsdteht, a suitable powder dlluer~t, suoh as lactose, and an optional surlbdartt, The ectfvs lngrodlent typioalty comprises from 0.~ to X40% wlw of the iotmufation. A second type of lltuitnwthro eet~otd generator vomprtsec a rhstArsd dose inhslsr.
Mot~rod dose inhalant an pr~au~'I~ed aet4sol dhpenssro, typioolly containing s vuspemion or aolutlan ib~rnulstlon of the active In~radienf an a Aqulfled propelisM. During use thtse devices dlscharpe the ibmuiletion throtrQh a valve adapts ~ air s metered volumr, typically from 'i 0 to 150 pl, to p~oduoe a Tlnepsrfk~ spray ovntalntnQ the active Z1 - ,...
.,~.-.._..gt 'd.~9~BL 'OlI S ; S a lI AY9~ ~8 BRRI 'f7 'Writ 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, s surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.
The aerosol, whether formed from solid or liquid particles, 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 fi0 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'' to about 10 '3 Moles/liter, and more preferably from about 10'6 to about 3 x 10'~ Moles/liter. Depending upon the solubility of the particular fomlulation of active compound administered, the daily dose may be divided among 20 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 2s 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 3o 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.

In administering the active compounds of the present invention, they may be administered separately (either concurrently or sequentially) or, alternatively and preferably, they may be pre-mixed and administered as preformed conjugates. As an illustrative example, as suitable dose of a s 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.
In the manufacture of a formulation according to the invention, active agents or the physiologically acceptable salts or free bases thereof o 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 ~s 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.
2o 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 25 dispersant which serves to facilitate the formation of an aerosol. A
suitable dispersant is lactose, which may be blended with the benzamil or phenamil in any suitable ratio (e.g., a 1 to 1 ratio by weight).
In summary, the transfer vectors of the present invention can be used to stably transfect either dividing or non-dividing cells, and stably so express a heterologous gene. Using 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.

Having now described the invention, the same will be illustrated with reference to certain examples which are included herein for illustration purposes only, and which are not intended to be limiting of the invention.

Models of Human Airway Epithelium 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 to 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 15 described (Wu, R., et al., 1985. Am. Rev. Respir. Dis. 132, 311-320), but omitting the filtration step. 1-2 x 106 cells are plated per 100mm tissue culture dish in modified LHC9 medium. Lechner, J. F. and Laveck, M. A.
1985. J. Tiss. Cull. Meth. 9, 43-48. The modifications include increasing the EGF concentration to 25nglml, adjusting the retinoic acid concentration 2o to 5x 10'6M, and supplementation with 0.5 mg/ml bovine serum albumin and 0.8% bovine pituitary extract. At approximately 75% confluence, the cells are harvested by trypsinization and passage 1 cells are plated at a density of 2.5x105 cells on Transwell-Col inserts (Corning-Costar, 24mm Qs, 0.4Nm pore size), in modified medium. The medium is similar to the 25 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. After the cells grow to confluence (4-6 days) the apical surface of the cultures are given an air-liquid interface for another 25-30 days until use.
3o Initial histological analyses of human WD cultures derived from non CF nasal airways after 34 days of culture indicate that the epithelium is pseudostratified mucociiiary, with abundant cilia and cell-types representative of those present in human nasal airways in vivo.

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~~ ~~. 1~ ~ JilN 1999 Esven Trsntrriernbratto ReQeptor Expntston on tho Api~l Merrrtrrane of WD Galls .
InveetiQaNo~ were eanied outto Idetd9f~ whloh 7-TM rocaptpra, if any, are fooal~ed fn the epleel membrane of WD human ainwy aplthafGal cells. Culturoa of WD calls wen axposrd to NECA (an Aa, receptor agvntat), Isaproteronol, brsuiyklnln, yr ATP (sill at l O~My, and Cl. seastory reeponelvenesa was determined. A: shown in l~tEuro S, a Ci- tecretory reepvnse was detested In thv presence o~ all of the egonisns, howswr, the qroataet response was observed In the presonoe of ATP. Thees results strongly eugpast that tut~ottona! adenoelns, ~-adron~gtc, btadyldnin and purino rsceptots sro proeont on the pptdsl surfeos et airway epithelia.
EXAMP~ 3 Bindbr~ and M~rt~allsation of AdV
in Hu~tt~n PD and WD ettwsy optthilial sells !=xporimsnts ehowlnn that e,denavtrue wcbor (/~dV~lnternadi~atbn.
not Adll-binding, !s the ribe~tlmltlr~ stop rpauiHnp In low ailldency ~sne transfior to RTE 1Np cutturos oro nspestod~~ with human arkuras to datermlne !t the same nste~.Ilmltlnp siJep Is prnant, if thls !s hideed ihs cast, then sltflough the oohs heave ~ raduc~ed rubs of Ins~ltranon, it rn.y be possible to Incroaee pane ~rsnil~sr oficlency to WG cuiiures by enhartr~np the amount of AdV that binds tO see cuidare~rpee, For a given concentnatlon of AdV, sxnoead tv ~dher PD or WD cuftunea, only approxirnstly 0,!-t 94 of the fate! AdV eaeposed to cavils remains attsohad slaver waehlne. Enhanaemant of AdV-blndinp stow that schivwed with s single aaposura taa~l8 tp rn incxuase In pohe transfer, since Increasing the blndlnd of AdV to veils will increase the pl~babtl'~ty thAt 4n Mtert~N~bon event Iw~da to AdV entry.
To determine the nltealmitfng slap fbr inetfiolent gins kanffer In human vultures, PD and YYD vunurrs era wcpovad by s°8.-I1d51lLacZ (1.2 x 101° p) for analyses of AdV.blndipp, ltredort and tranagane ~Ri '~ ~aRl, vu t ~ s a a rwAf:a ~aRr ~r~ v~~

expression in the human cultures. To investigate the effect of increasing the concentration of AdV and/or the duration of exposure to AdV, PD and WD cultures are exposed to 35S-AdSVLacZ {Pickles, R. J., et al., 1996.
Human Gene Therapy 7, 921-931 ) at a range of concentrations (10'-10'2 plml) for a number of time points (1-24hrs) at 4°C, after which cultures are washed in medium and then divided into three groups for analyses.
Binding is measured as cell-associated radioactivity. internalization of bound AdV is measured by transferring the cultures to 37°C for 6 hrs followed by measurement of cell-associated radioactivity after removal of non-internalized radioactivity. Expression is measured by transferring the cultures to 37°C for 48 hrs before measuring a-gal activity.
Radioactive counts per minute (CPM) and ~3-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, internalization and gene expression as tong as saturation of receptor uptake and expression systems does not occur. With WD
2o cultures, although a 10-fold increase in AdV attachment is expected, the corresponding 10-fold increases in internalization and expression are not.
These data indicate that increased binding alone does not overcome the rate-limiting step (internalization) into WD cultures.

Targeting of AdV Vectors to the P2Y2 Receptor To test the concept that the P2Y2 receptor is a candidate receptor for targeting based on the ability to bind and internalize an exogenous ligand, we have obtained CHO and A9 cells (both of which are not so transducible by AdV) that express the HA-tagged human P2Y2 receptor (HA tag on the extracelluiar N-terminus) by retroviral gene transfer. HA-P2Y2 receptor expressing A9 cells, but not control cells, stain with fiuorescently labeled anti-HA Abs under resting conditions. With agonist CA 02295315 1999-12-15 gIMB_A5-~
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~- ~ ~~ ~ $ Jt~~ X99 (ATPrs (1 ~ Mu axpaauro, approxinn~ly g0~6 of tho roooptors are ir~brnr~llssd wnhln 45 minutes. Ths IntemalJzadon of PZY~ reoepmr is madlatad vla aortod pits.
Next, a bi-opeoHto antibody (bs~) tpproe~oh v~g used tv east whethor P2Y~ raclp~or could modlata 0~ne transtor. Ardlbody HA.11 (BabtO) aps~lns~t ~fluerura hsmaaplutln (snti.HA) is dt~obed a~alnst the HA-Apltvpe innrtsd into en extracellular domain of the human P~Yx receptor vrfilch b oxpnavd In 18Z1N1 human estrocyboma astls. The brid0lng antibody is produ0ld by rstCdng an ptllf-flbsr (knob) antibody with rr~MeleknldoDsnt~yl-N-hydn~oqlsuliatuodlnlmld~ s~ (Sutfo-MBS, pierce, Rodcford, It.) or N-(~alalmidobutyryloxY) tulfoauodnihlido e~itsr (sutlb-GM~8, Pforoe. l~eldvc~, It-) a! neub~f pN. Aftor roduction of anti-HA by rn,~aptoethylamfna, tnd deealttnp. the two evtlbodfes are mixed.
enablltt~ disuttde Ct~ost-link folmstion, Bt-functional antibody is putMod by soqua~lal chromttopraphy aver fiber pn~toln and HA ootumns.
Ustrt~ thl! brAbs Itdainst the Ad Itbor (itnob) and the HA oplLvpo tai (alqbar x aliA), blndlr~ of btrAb and AdV to HA-PxY~ ncoptor, but not to null eocprsatnp A9 ceth has been dsmonstrsted. AdV bound to A9,HA-P2Ys oelte tn the pressrtoo, but not fhs aber~ttos, of bl~Ab.
Mdre Impo~tMly, PZYg rovaptor spaaflc Oohs transfer in AS and CHO oslh has bostt tchieved ulinp tho bt~Ab approach. In one protaool omp~Yl~~ (1 ) ~q~$I eapoeura at a~C of HA P2Y~ racoptor exp~tlnQ A9.vsnus null vecttir~xpr~bnit~ Ag c:allt to varyiap oonoentrsrtioni of bs~ldt (anb-HAltntl-fiber knob; aroduaed by Dr. R.
Piddes in the lsbotl~bory of Dr. D. 8~t ~ttt~e NtH) i~ollow4d by IIdV~IaoZ
(psrtldes 01~1 d~ (PlcWss, R. J,. eat al.,1908. Hurruen Gene. Therapy y 921 ~Q31 ): (2) Inwballon fbr 1 hour at 3TC with bpanist (A~TF~S.10'~ M), f~dla0~wod by Incubatbn fbr 24 hour in madlwm: and (3) quandtatton of Qane tr~nsfsr afAolency by oountlng tnd oalculatmp the percent IacZ potttJvo oolis, tt was oburved that HA- P~Yz reoaptor oxpnstinp A9 aolls aro tranrdeosd by I~d-tacZ e~ s function a!ttho conosntratton of b~mAb.
wltarrae nuU A6 cells sro not (Fi~uro 1A). Indeed, It appsan that noariy 1 On9i pena transhr attlciericy is approacttod wlih 30 wylrnl of bs-Abe.
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'!n a second protocat using preformed conjugates, aup~~Mat~on of gene tremaller thAt peaked et ~90 pplml b~Ab waa observed (Flouro ~A).
The fail In tranaduction efNclency at higher bs-Ab vonCentr~ora may n~iect oompetiteon by unbound be.Ab ibr the tar~e~t. .
similar lncrw~ess tn Qene transfer to HA. P2YrR A9 cells with be.
Aba against h!A snd biotin (aHAJabiobn~ and blotinylstsd ad.rtovirat we:Evre, Finally, the tpeolnolty of the pane trend~r observed with bs~Ab in Ag~HArP2Ya-R cells we evaluated (Plaure 48). HA- P2Ya~A9 cstl~
exposed to Ad-L~acZ anQ A"i'Pr5 =howsd almost 140'6 exprwsivn oZ t,,acz.
I~ ~t~tet~ almost no Lack sapnaaion was observed with an Irreiewnt bs-Ab (ltltt!-ftd9nti,AD P1 ~Itoply~ nor In the prosenol 0f a 40X sx~deas of !?ree anti-HA sraabody, nor In calls that had been Pre-exposed ibr 24 hour to a high oonCentnt~tvn oispvnist hereby inducfnp c~tl.~~ ~~p~r down,reQuhrtton~, 'These data eslsblish thst the NA~P2Yz recep~r medtstea gene tranei~ir v1a epsClllo In:~tior~s with artd-HA bs.Ab:.
BXAMPIE ~
Exploitation of Other CeilulAr Uplake Msahanlame to increase AdV~enty Into YYD Ct~lturee The e~lfsc~venera of pens transfer by tarpe~n~ AdV ~p recepEora that undergo tntenwll~ion la dope~ent on the irrtemalliration efid~cy o! the receptor. To lest tt~e inbemaltzotion e!!ldenoy of P2Ys-reoaptcrs, a hurnrn P2Yrroceptor (~ri an tr~f(cisnsa hentaQlubfn (HA) oplEopv-tag inserted inbo the extreu~nuler damatn) hay bevn over~cpreaaod In 1~2i N1 human astrooytapma ostt:, S. aromek srtd T, K. Harden, Molsauisr Phannecotopy 64, d4:4B5 (t sae). P2Y~-HA expreatinp cells were tnc:ubstsd st 3T°C v~th a non-hydroiyzabte ATP ana>aAua, ATPy9. At specMc llrne ,point:, after apvn~t ~tddluon, the cells were ~~ wkt,aut penneablllaatlon In 4~6 petactormaldahyde and west. Monoclonal anti.
HA arnibody wee inarbat~ed with the cells ft~lowe~ by fncubedon with Cy3.
~nlugarid goat pnli~mouee tQ0 secondary aMrbody (Jackson tmrnuno ~s~rch Labs). 1'he eva0ablltty of P2Y~HA receptors to the and-HA wsa vfeuaitred with a tlaoheosnt m~cro~roope. The aatrooyboma calls wore 1i-t~ . ~$, V
.. .~.,-. __t~ ',~gGRI' 'dN S 9 S 8 R tIItRC:R RRR1 'r7 'Nnr fixed and immunostained for the presence of P2Y2-HA-receptor in the absence of ATPpS and after 30 min ATPyS exposure. In the continued presence of agonist there is a clear loss of immunoreactivity from the plasma membrane and the punctate fluorescent signals, indicating sequestration into endosomes. With ELISA assays internalization was quantitated to occur with an efficiency of 80% (loss of receptor sites with ATPyS exposures of 1 hr, Dr. Ken Harden, personal communication). This indicates that in the astrocytoma cell-line, internalization of activated receptors occurs efficiently.

Gene Transfer Across the Apical membrane of Polarized Epithelium Confluent MDCK renal cells were used as a model of a polarized, AdV-resistant epithelium. In MDCK cells expressing the HA-P2Y2 receptor, 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-P2Y2 receptors in the apical membrane of polarized MDCK cells desensitizes, in part, by intemaiization of receptors. Bs-Abs directed to 2o HA- P2Y2 receptors sequentially administered with AdV-GFP (green fluorescent protein) and ATPyS transduce HA- P2Y2 receptor, as compared with Neo-expressing MDCK cells. Thus HA-P2Y2 receptor specific gene transfer has been achieved in polarized, AdV-resistant cells.

Gene Transfer in A9 Cells Expressing the Bradykinin II (B2K) Receptor and in CHO Cells Expressing the P2Y2 or (i2 Receptors The general applicability of the above approach has been 3o demonstrated in other cell types and with other receptors.
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 SENT BY:SIMBAS ~ ~_25_pu ~0~2 j~rms~ 1999-i2-is 5IM8'S~ f~ ~ ~ ~~ i ~ ~ r$ ~~3 ' ~~ 1~ J UN X999 bratdykinln. Briafly, aelle st 4°C were Incubated In the ibrenoe or proaenos of bfipr~c antibody (ba~Ab, l0irglmt fbr 2 hrs), washed and exposed bo AdVLacZ (1 t1s° pselides far 2 hra?, weshad and exposed to bradytdnln (SK,1 yIM for 2 hn at 3?°C). The celte were Irian mairtteined at 37°C ford hrs undl grne vxpneslon woo asese~svd by standard teehnlques. rll~4ln 3 shows that a~fllr~ant qenv expression occurs only in HA82k-expraasit~p cell: Ineub~d with botfi bs-Ab and AdV with enhaneemvnt of gen* transior by activa~irt~ the naovptar with aaonlat.
Alrrb~tt no vxpreelJon was abr~nrod with AdV alone, and only modest levete ware obsenhd in the prosenoa otAdV t bf-Ab (indivating low-Ivval rsosptcrtum-ovrr~ even fn the abasncv of i'~pand). Only negllpiblv t.~Z
vxpnee~afon was obsena~ in null A9 cells re~t~dles~ of tTertrr~arit.
In additional studies, CHO oeiis (Chinese Wamatsr Ovary cello whk~1 iedt the AdV atlachmertt ~soapbor) Iftpreaslng 1hv HAwpltopo tapqvd ~-adretlansvaptor afld wRd~yPa (Vltt) CMO cells wets ~raquvr~tially expoa~! ~
InasaelnQ oonosnthtlons o~( bf-l~sdfiC antibodloa (~ttKl-fibt~rta~ob x ardl-HA) and AdV and flnaAy ~ isaprv~ronot. 9~ie~lly, ~s st 4°G were Inarbabad tn the absanve or prssvnoa o! b?'speclflc antibody (bi-Ab, f?,1-l0~lml for Z hn), wevshad and vxposad to Adll~cx (10'° pattkiea for 2 hn), washed and exposed to i=opraterenol (iONM for 2 nrs at 3T°C). The mile ate then metr~talnod at 37°C for 24 hr~ until gvrw vxprasaion wAa anoaed by denalbometry of L»C.~ expressing t;sslla shown as arbitrary unite. RIgWrv dA~l~ ahvwa ba-Ab dode~deprndsnt tncrorsaa in bane expresaJon only In CHO orll: vxpreseinq HA.PZYrR ~l~uro ~A) cr HArpz {Fl~un sA) rec~ori.
~XAI~APLE g Biotteylfatad ~l~oniale Mother appr~Owd~ tv taroet vactan lei the P2Y~ roaeptpr (or any ether 7-TM raoepto~ 11 by ch8rriical linkage to s tnddifled apontatlantaponiat mole0uls es s tarpvt molecule. A prot4~rpi e~ponht-iir~kvr la bbbn (B~.UTp, which oor>roatns a 16 carbon linker oonneofina the 6 posittlon o~f the pyrimldlne bees to biotin, 8-UTP is an a9onlot of PAY=
_ . . so .._.,..,_,..~.aa 'a.. 996L'0~ s ~ s a ~ ~vsE:o 8s6c 'EZ W

SENT BY:SIMBAS ~ CA 0229s31s 1999-i2-is gIMBA~~ 819 953 ~5r38~14 1-25-Gu m c~3irm , ~~ jy~ ~ ~ I ~. 7 ~. ~~ IS Jt~~1 '199 ors (Flpure r). Flu4teeoones studtea have dl~montb~abd that when CHO oetts exptssslnp P2Y,~n~ptoro aro sxpOeed to B~UTP ooryupsted to strs~vidin Texss Rid (TR), the 9-IJTP trippehs and is i~ernaliaed wttt~
the PZYa tscep~ore. .
In a RWhar study, pens tar~cttnp by 8-UTP was evaluated In Aq tills sxprw~sinp 1~IA~P2Ys r~sceptvrs. HA~P~lfs-AD cells were sequentially aucposed to BrUTP, stnptavldln (SA). and B~AD (blaMn labetsd adenovirus a~cprssetnp LacZ). Approximately, ~0% of NA~P~Y~ cells demonstrated LecZ expASllon (>f:lyure 8). In corstr's~t, only Iow levels of ~saZ sxpreesion were obsdrwd in wtld-type A9 ostls, , i ~7UMPLp p Dlnuc!~atlde A~ontetr Non-hydroly:~bie bidoplcs4y aatlvr enrlops of UTP 1br Ilnicape to vectors have been developed. 'fhe dinucl4oNdo UePi his idea! properdes.
As shown In l~t~uro 8. U~P~Is equ'~pvro~tWith UTp in IhdudnQ a btolvplcal refponse (inoMlfol Phosphate csleate) end is ra~rtstar~ to hydrolysis In aysbc fibroNs (CP) sputum.
. pXAMPLE :10 Inwstlpetlon Into tho AdV-Inboras~bon processes tn Humen PD wnd IIYD Gulturos The aw~3ee l~0dns aro ~epor~d to rnvdlaf~s i~emall:~tion but not ettechmant o1 Wd lr~bo splthelio) cells !n vitro. Tha foc~lizotivn of the ~ .
membrane-bvurut acv~~ tnboprtns to the aplasl endl0r bt~e~el mvrnbrsvea of WD wlturss Is aruClal In ur~dsrnta~tdlt'~ the rolaa of these molevules in AdV~mvdisted asps trsnaibr. The avsliabfllfy et a number of different ar~lbodies to these Inteprino ello~ws their lochttan in polar~xed sp>thslla~ ~o be detemnlned. The Inteptfi~ ere also teospton for pep~des oontainlnp R~3D amino add sequences. A number o! studlrs havo shown thst RGD~pspt~s inhibit AdV-mediated gene trsnsf~ tv epnhellal cells by irrbsraction with the acv~aa inte~rlns. This i"rxample mustrates the efl~ct~r of __ .......... , _ .. 31 S 'i S 61I RVB~ ~6 6fi6t 'Et 'ttal . _EZ a.--...9S6t O1~L,.,._ .

SENT BY:SIM8AS ~1_Z5-OpCF;~~239~m1s 1999-~2-~s SIMBAS-~ 819 953 9538;#15 w . '~If~iy~Jl3ss b ' .., ,.~, ~.~s JUN 1'999 ROD piptldee cn AdV-binding. t~rmxilzador~ and trtnoQsne axprebeton in PD ~tt~d WD cuiatr~ls.
An tnteprfn u~tibody~spr~ia lmmurropr~ecipttation procedure has been developed tnltlally with RTE WD caftans to Iocallze the ay~iye .
ir~beprin;. 8inos tJ~ttbodia to these Intprfna are not admmsrc>olly available, ws hws obtained en antibody (R93a. a kind Qitt irvm Dr. 8~von Atbelda. Unfv. of Penn, PA). reined a~pafnst the human endothelial ref!
vttronectln receptdor whloh has barn lbu red to cross-reed with rst aw~a U~teptine is used, BrieMy. etwar the apical or basotatetat dor~tns of NVD
cultures were exposed to 9uito-NH8-9iottn (0.3 mglmt, Pteroe) et ~1°C
to blOtinytabe only sxfernat membrane pro~talM, After soiubttizatlon of the oelle In a nondsnaturfnp iysls buffer (in th~ preasnoe o! protease ~hlbltrari). proteins we~a Immunaproctpftabsd and ssperoted by Western analysis on a 4~1~% avryiamtda gel (Noveut) under non.~edudng condidona. The bloanytatsd pro%tno wvro probed with rUepdwtdin-co~r~ugatod pdroxidaH eooondary antibody and detecaed by JCL analysis (Supersbnai C~.~f~IRP, Pivrct). Tho btolMylat4d protslru idsnti~wd by Rd38 ore shown in ft~urv 6 (bands at l~SkD oorrospond bo the av and 97kD sr~e p~ ~eubunitl~nd appear fio bo pfdserst in the basolatat'tl membranes of RTE oelis and 1n hlvLa oetis but absent from the apical mernbtatne ofithe WD cutiuns, w0gsssinp that rat vtbonectfn receptors may not be fooatsd apicalty In these oul~rs-tY~ss. 'rhe at»encs of these ,.
intsprlns M tht sp'~oa1 mare~bnns oouid eaoount fior the low rato of AdV-lntrsmatlzation Into these vultures. ~ , eXAMPL.~ i ~
IdrntMcatton and Locaftxation of inleprlna present on Human PD and 1ND Cuttuns Protsjne from firs sptcal andlor bae~iaberal membranes of human Pp and WD cutbrros Art ssledtvety tsototed by exposing Mdlvldusl surietces to Sulpho~NH8~Btotln at ~°C. Standard inlmunopreotpitatlons experiment srs ps~rmed with seltvdve human errllbcdlee (LM808, uv~°
P1 Fo, wvps i Vt~R'f~f7, ay ~ P4G11; pt : C06~. ~~ i anti-~6 and RB3S, !Z ' "'' 9 q ~ RYOt ~ 6 686 t vZ vflr ... ._~a 'a-_e5sl aH

av~33i5 ; all obtained from Chemicon Inc, CA.). This procedure allows for detection and localization of the av~3 integrins to the apical and/or basolateral membrane.

Interference of AdV-Internalization by RGD Peptides.
Adenoviral attachment, internalization and transgene expression in PD and WD cultures will be measured, as described above, in the absence and presence of RGD-peptides. Hexa-peptides, the bioactive o GRGDSP (Gibco-BRL) and the inactive control peptide GRGESP (Gibco-BRL) will be 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'° p/ml).
Cyclical RGD peptides (Immunodynamics, La Jolla, CA.), reported to be more potent at reducing AdV-mediated gene transfer, are also used.
~5 Analyses of AdV-attachment, internalization and transgene expression are performed as described above.

Investigation of the Cellular Uptafce Processes for AdV-entry 2o Initial attachment of AdV to epithelial cells occurs via the fiber (knob) protein. It is unclear whether fiber protein alone is sufficient to trigger internalization and endosome formation or whether the role of fiber is to aid the virus to locate and exploit an inherent endocytotic event.
Internalization of AdV into the cytoplasm however, is mediated, in part, by 25 av~i3~5 integrins. T. J. Wickham et al., Cell 73, 309-319 (1993). It has been speculated that av~i~,5 integrins are absent or low in number in the apical membranes of both WD cultures and cartilaginous airway epithelium (M. J.
Goldman et al, J. Virol. 69, 5951-5958 (1995)), possibly resulting in both the low rate of internalization and gene transfer efficiency in these cell-ao types. Therefore, the potential cellular uptake processes that may be responsible for entry of AdV into cells are investigated. First, to understand the functional role of the fiber (knob) protein-cell interaction, we will conjugate knob protein to fluorescent microspheres of the same diameter as Ad and assess the initial interactions of the knob-spheres on PD and WD cells by confocal microscopy. We will specifically test the hypothesis that increased specific or non-specific binding can increase internalization and possibly expression. Second, we will overexpress human av~35 integrin in WD cultures to direct this protein to the apical membrane. If this is feasible, we will test the hypothesis that av~35 expression on the apical membrane is the rate-limiting step for internalization and hence gene expression. Third, in order to understand the cell-entry pathways utilized by Ad, we will investigate cell-lines that are either deficient or competent at coated pit receptor-mediated endocytosis.
We will test the hypothesis that high concentrations of AdV may use non-specific entry pathways to gain access into cells. Finally, a strategy will be tested, as an experimental concept, to increase the internalization efficiency of AdV into WD cells by exploitation of other cellular uptake s mechanisms, i.e., targeting AdV to specific receptor types that undergo endocytosis when stimulated by exogenous iigands.

AdV Internalization into HeLa Cell-Lines 2o 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. R. M. Steinman et al., J. Cell Biol.
96, 1-27. We will study the role of coated-pit receptor-mediated 2s endocytosis and non-specific pinocytosis on AdV-entry into cell-lines which have either competent or defective receptor-mediated endocytosis. 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). H. Damke et al., J. Cetl Biol. 127, 915-934. Normally, dynamin 3o 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) fait to be internalized into cells expressing mDyn, but ligand-receptor binding, coated pit s assembly, recruitment 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.

Uptake Processes for AdV-entry into HeLa Cells.
We will use HeLa cells either overexpressing wild-type or mDyn (gift of Sandra Schmid, Scripps Research Institute, La Jolla, CA) to study the uptake processes that are prevalent for AdV-entry into these cells. A
range of AdV concentrations will be studied to determine if high titre AdV
leads to cell-uptake by non-specific processes. Briefly, monolayers of mutant HeLa cells, grown on plastic, expressing wild-type or mdyn dynamin will be exposed to AdSVLacZ (106-10" i.u.lml, corresponding to 2o an MOI-1-105) at 4°C for 2hrs and then transferred, without washing, to 37°C for time-periods of 0-24hr, at which point the cells will be washed and maintained at 37°C for 48 hrs before ~3-gal enzymatic assays are performed. Differences in the gene expression observed in the two cell-lines at specific time-points will reflect the participation of receptor-mediated endocytosis on AdV-entry. In conjunction, we will perform comparative studies with fluorescent microspheres (with and without attached knob-protein) to delineate the interaction of these proteins with specific and non-specific uptake processes.

WO 99/00511 PC'T/US98/13336 Exploitation of Other Cellular Uptake Mechanisms to Increase AdV-entry into WD Cultures The effectiveness of gene transfer by targeting AdV to receptors that undergo internalization is dependent on the internalization efficiency of the receptor. To test the internalization efficiency of P2Y2-receptors, a human P2Y2-receptor (with an influenza hemagluttin (HA) epitope-tag inserted into the extracellular domain) has been overexpressed in 1321 N1 human astrocytoma cells. P2Y2-HA expressing cells were incubated at ~0 37°C with a non-hydrolyzable ATP analogue, ATPyS. At specific time points, after agonist addition, the cells were fixed without pem~eabilization 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).
~5 The availability of P2Y2-HA receptors to the anti-HA was visualized with a fluorescent microscope. Cells were fixed and immunostained for the presence of P2Y2-HA-receptor either a} in the absence of ATPyS, or b) after 30 min ATPyS exposure. In the continued presence of agonist there is a clear loss of immunoreactivity from the plasma membrane and the 2o punctate fluorescent signals, indicating sequestration into endosomes.
With ELISA assays internalization was quantitated to occur with an efficiency of 80% (loss of receptor sites with ATPyS exposures of 1 hr, Dr.
Ken Harden, personal communication). This indicates that in the astrocytoma cell-tine, internalization of activated receptors occurs 25 efficiently.

Analysis of Transgene Expression in Human PD and WD Cultures PD and WD cultures are exposed to AdSVLacZ by application to 3o either the apical andlor basolateral membranes over a range of viral titres (106-10" infectious unitslml: corresponding to MOI range of 1-105) 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 internalization. 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 Therapy7, 921-931.
While the invention has been described in connection with specific embodiments thereof, it will be understood that the invention is capable of further modification. This application is intended to encompass any variations, uses or adaptations of the invention that follow in general, the principles of the present invention and including such departures from the present disclosure as come within known , or customary practice within the art to which the invention pertains, as may be applied to the essential features set forth in the scope of the scope of the embodiment of the invention described above.

Claims (65)

THAT WHICH IS CLAIMED:

1. A method of delivering a heterologous nucleic acid into a cell, comprising:
contacting a conjugate to said cell, said conjugate comprising a transfer vector and a ligand, wherein said transfer vector comprise a heterologous nucleic acid to be delivered into said cell, and wherein said ligand specifically binds to a G protein-coupled receptor, and wherein said cell expresses said G protein-coupled receptor, under conditions that causes said vector to be internalized into said cell.

2. A method according to claim 1, wherein said vector is a viral vector.

3. A method according to claim 1, wherein said vector is a viral vector selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, human retrovirus vectors, nonhuman retrovirus vectors, and herpes virus vectors.

4. A method according to claim 3, wherein said viral vector is selected from the group consisting of lentivirus vectors and Moloney Murine Leukemia virus vectors.

5. A method according to claim 1, wherein said vector is an oligonucleotide.

6. A method according to claim 1, wherein said ligand is an antibody.

7. A method according to claim 1, wherein said ligands is a peptide.

8. A method according to claim 1, wherein said ligand is selected from the group consisting of nucleotides, nucleosides, ~~techolamines, C~A, and bradyk~nin.

9.~A method according to claim 1, wherein said ligand is selected from the group consisting of G protein-coupled receptor agonists and G
protein-coupled receptor antagonists.

10.~A method according to claim 1, wherein said conjugate is a convalent conjugate.

11. A method according to claim 1, wherein said cell is an airway epithelial cell.

12. A method according to claim 1, wherein said cell is a differentiated columnar airway apithelial cell.

13. A method according to claim 1, wherein said contacting step is carried out in vitro.

14. A method according to claim 1, wherein said contacting step is carried out in vivo.

15. A method according to claim 1, wherein said conjugate is formed prior to said contacting step.

16. A bispecific antibody having a first combining region that specifically binds to a viral vector and a second combining region that specifically binds to an extracellular epitope of a G protein-coupled receptor.

17. A conjugate useful for delivering a heterologous nucleic acid into a cell, said conjugate comprising a transfer vector and a ligand, wherein said transfer vector comprises a heterologous nucleic acid to be delivered into said cell, and wherein said ligand specifically binds to a G
protein-coupled receptor.

18. A conjugate according to claim 17, wherein said vector is a viral vector.

19. A conjugate according to claim 17, wherein said vector is a viral vector selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, human retrovirus retrovirus vectors, nonhuman retrovirus vectors, and herpes virus vectors.

20. A conjugate according to claim 17, wherein said vector is a viral vector selected from the group consisting of lentivirus vectors and Moloney Murine Leukemia Virus vectors.

21. A conjugate according to claim 17, wherein said ligand is an antibody.

22. A conjugate according to claim 17, wherein said ligand is a peptide.

23. A conjugate according to claim 17, wherein said ligand is selected from the group consisting of nucleotides, nucleosides, cat~cholamines, C~A, and bradykinin.

24. A conjugate according to claim 17, wherein said ligand is selected from the group consisting of G protein-coupled receptor agonists and G protein-coupled receptor antagonists.

25. A conjugate according to claim 17, wherein said conjugate is a covalent conjugate.

26. A method according to claim 3, wherein said vector is an adenovirus vector.

27. A method according to claim 8, wherein said ligand is bradykinin.

28. A method according to claim 8, wherein said ligand is UTP or an analog or derivative thereof.

29. A method according to claim 28, wherein said ligand is U2P4.

30. A method according to claim 1, wherein said ligand is expressed in the adenovirus knob protein.

31. A method according to claim 1, wherein said G protein-coupled receptor is selected from the group consisting of dopamine receptors, muscarinio choligergic receptors, .alpha.-adrenergic receptors, op~ate receptors, cannabinoid receptors, ~~rotonin receptors, .beta.-adronergic receptors, purinoceptors, and the C~A complement receptor.

32. A method according to claim 1, wherein said G protein-coupled receptors is the bradykinin receptor.

33. A method according to claim 1, wherein said G protein-coupled receptor is the P2Y~ receptor.

34. A method according to claim 1, wherein said cell is a human cell.

35. A method according to claim 1, wherein said heterologous nucleic acid encodes a therapeutic protein or peptide.

36. A method according to claim 1, wherein said heterologous nucleic acid encodes an immunogenic protein or peptide.

37. A method according to claim 1, wherein said heterologous nucleic acid encodes the cystic fibrosis transmembrane conductance regulator protein or a biologically active analog, fragment or derivative thereof.

38. A method according to claim 1, wherein said heterologous nucleic acid encodes antisense sequence.

39. A bispecific antibody according to claim 16, wherein said viral vector is selected from the group consisting of adanovirus vectors, adeno-associated virus vectors, human retrovirus vectors, nonhuman retrovirus vectors, and herpes virus vectors.

40. A bispecific anthibody according to claim 39, wherein said viral vector is selected from the group consisting of lentivirus vectors and Moloney Murine Leukemia virus vectors.

41. A bispecific antibody according to claim 39, wherein said viral vector is an adenovirus vector.

42. A bispecific antibody according to claim 16, wherein said G
protein-coupled receptor is selected from the group consisting of dopamine receptor, musc~rinic cholinergic receptors, .alpha.-adrenergio receptors, opiate receptors, cannabinoid receptors, serotonin receptors, .beta.-adrenergic receptors, purinocaptors, and the C5A complement receptors.

43. A bispecific antibody according to claim 42, wherein said G
protein-coupled receptor to a bradykinin receptor.

44. A bispecific antibody according to claim 42, wherein said G
protein-coupled receptor is the P2Y2 receptor.

45. A bispecific antibody according to claim 16, wherein said bispecific antibody comprises a monoclonal antibody.

48. A bispecific antibody according to claim 46, wherein said bispecific antibody comprises a monoclonal antibody directed against the adenovirus fiber protein.

47. A conjugate according to claim 19, wherein said vector is an adenovirus vector.

48. A conjugate according to claim 23, wherein said ligand is bradykinin.

49. A conjugate according to claim 23, wherein the ligand is UTP or an analog or derivative thereof.

50. A conjugate according to claim 49, wherein said ligand is U~P4.

51. A conjugate according to claim 17, wherein said ligand is expressed in the adenovirus knob protein.

52. A conjugate according to claim 17, wherein said G protein-coupled receptor is selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, .alpha.-adrenergic receptors, opiate receptors, cannabinoid receptors, serotonin receptors, .beta.-adrenergic receptors, purinoceptors, and the C5A complement receptor.

53. A conjugate according to claim 17, wherein said G protein-coupled receptor is the bradykinin receptor.

54. A conjugate according to claim 17, wherein said G protein-coupled receptor is the P2Y~ receptor.

55. A pharmaceutical formulation comprising the conjugate of claim 17 in a pharmaceutically acceptable carrier.

56. A method of delivering a heterologous nucleic acid into a polarized epithelial cell, comprising:

contacting a conjugate to said polarized epithelial cell, said conjugate comprising a transfer vector and a ligand, wherein said transfer vector comprises a heterologous nucleic acid to be delivered into said cell, and wherein said ligand specifically blinds to a receptor on the apical surface of said polarized epithelial cell.

57. A method according to claim 56, wherein said polarized opithelial cell is a polarized airway epithelial cell.

58. A method according to claim 58, wherein said polarized epithelial cell is a polarized kidney epithelial cell.

59. A method of administering a nucleic acid to a subject, comprising:
administering a conjugate to said subject, said conjugate comprising a transfer vector and a ligand, wherein said transfer vector comprises a heterologous nucleic acid, and wherein said ligand specifically binds to a G protein-coupled receptor, under conditions that said conjugate binds to a G protein-coupled receptor expressed by a cell and said vector is internalized into said cell.

60. A Method according to claim 59, wherein said subject is a human subject.

61. A method according to claim 60, wherein said subject has cystic fibrosis.

62. A method according to claim 59, wherein said conjugate is administered to said subject by a route selected from the group consisting of oral, rectal, transmucosal, topical, intestinal, inhalation, intravenous, intramuscular, subcutaneous, intramedul~ary, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intra~cular administration,

63. A method according to claim 59, wherein said conjugate is administered to the lungs.

64. A method according to claim 59, wherein said G
protein-coupled receptor is expressed on the apical surface of a polarized epithelial cell.

65. A method according to claim 64, wherein said polarized epithelial cell is a polarized airway epithelial cell.