JP2002533110A - Introduction of target gene using G protein-coupled receptor - Google Patents

Abstract

(57) Abstract: methods of delivering heterologous nucleic acid (e.g., gene sequence) into cells, the virus comprising the heterologous gene sequence, is attached to G-protein coupled receptor (i.e., 7 cell transmembrane receptors, such as P2Y ₂ receptor) Including. The virus can be attached by a cross-linking antibody or by the binding of an antibody specific for the receptor and an antibody specific for the virus, wherein the antibody that specifically binds to the receptor and the antibody that specifically binds to the virus are Crosslinked. Alternatively, the virus may express a peptide that specifically binds to the receptor. Receptors can be induced and internalized by the addition of a ligand known to cause internalization of the receptor into cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This invention was made with government support under Grant No. HL51818 of the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION [0002] The present invention relates to methods and systems useful for introducing nucleic acids into eukaryotic cells.

BACKGROUND OF THE INVENTION The ability to introduce certain foreign or native gene sequences into mammalian cells and to regulate the expression of that gene is of considerable value in medical and biological research. Such functions provide a means for studying gene regulation and designing the therapeutic basis for disease treatment.

[0004] Introduction of certain foreign or native genes into mammalian host cells is facilitated by first introducing the gene sequence into a suitable nucleic acid vector. Various methods have been developed that allow such recombinant vectors to be introduced into desired host cells. Use of viral vectors allows the recombinant molecules to be rapidly introduced into a wide variety of host cells. In particular, viral vectors have been used to increase the efficiency of introducing a recombinant nucleic acid vector into a host cell. The virus used as a vector for transduction and expression of foreign genes in mammalian cells is SV40.
Viruses (see, for example, H. Okayama et al., Molec. Cell. Biol.
5, 1136-1142 (1985)); bovine papilloma virus (eg, D. DiMaio et al., Proc. Natl. Acad. Sci. USA 79).
, 4030-4034 (1982)); adenoviruses (for example, JE.
Morin et al., Proc. Natl. Acad. Sci. USA 84, 462
6 (1987)), adeno-associated virus (AAV; see, for example, N. Muzyc).
Zka et al. Clin. Invest. 94, 1351 (1994)),
Herpes simplex virus (eg, AI Geller et al., Science 2
41, 1667 (1988)), and others.

[0005] Efforts to introduce recombinant molecules into mammalian cells have been hampered by the inability of many cells to infect the viral or retroviral vectors described above.
Limitations with retroviral vectors include, for example, the relatively limited range of hosts, based in part on the expression levels of membrane proteins that act as viral receptors. M. P. Kavanaugh et al., Proc. Natl
. Acad. Sci. USA 91, 7071-7075 (1994).

[0006] Accordingly, there is a need in the art for improved methods of introducing and expressing genes in target cells.

SUMMARY OF THE INVENTION [0007] The disadvantages of current methods of introducing receptor-mediated genes are overcome by the methods and complexes of the present invention. In particular, the present invention provides that the rate-limiting step of uptake of a viral vector by a cell surface receptor is not the event of binding the virus to the receptor, as was originally thought, but rather the internalization of the receptor itself (integer).
rnalization) based on an unexpected finding by the inventor. Accordingly, the present invention relates to novel complexes that facilitate the introduction of nucleic acids into eukaryotic cells. The present invention allows for the targeting of transfer vectors to specific cell types, the attachment of vectors to cells, and the regulated cellular internalization of vectors.

[0008] The present invention involves the binding of a transfer vector to a receptor that is internalized by a cell. Receptors are those that are internalized by the cell when the cell is exposed to a specific ligand, ie, that the receptor may be induced to internalize by exposure to such ligand.

DETAILED DESCRIPTION OF THE INVENTION The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. However, the present invention may 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 the specification.

[0010] The transfer vector receptor complex of the present invention comprises
Includes transfer vectors linked to receptors that can internalize cells. Transfer vectors can include foreign nucleic acid sequences (eg, genes) and can express foreign proteins or peptides. In a particularly preferred embodiment, described in more detail hereinbelow, the transfer vector uses an antibody specific for the receptor or a peptide expressed by the transfer vector that specifically binds to said receptor. Or using natural or modified ligands to target seven-transmembrane (7-TM) receptors. The transfer vector can be any suitable vector, including viral vectors, plasmids, oligonucleotides, or RNA / DNA chimeric molecules, as described more fully herein below. Interaction between the 7-TM receptor and the target complex results in receptor-complex internalization, whereby the heterologous nucleic acid carried by the transfer vector is introduced into the cell and expressed there.

A. Viral Transfer Vector One embodiment of the present invention is described with reference to FIG. In this specific example,
The complex (or conjugate) of the invention comprises a viral vector 10 (shown as an adenovirus in the figure) attached to a 7-TM receptor 20, which receptor 20 is present on the cell surface 100. The viral vector 10
Bifunctional bridging antibodies
ody) 30 to attach to the 7-TM receptor 20. Bifunctional cross-linked antibody 3
0 consists of one antibody 40 that specifically binds to the viral vector. Antibody 40
Binds chemically to an antibody 50 that specifically binds to the 7-TM receptor 20.

Although the viral vector 10 is shown as an adenovirus vector (AdV), the present invention relates to human and non-human retrovirus (ie, molony and lentivirus such as Moloney murine leukemia virus) vectors, adeno-associated It will be appreciated that other viral vectors, including but not limited to viral (AAV) vectors and herpes virus vectors, can be used to perform (FIG. 2). A viral vector of the invention can be an attenuated virus or can be rendered non-replicating by any method known to those of skill in the art.

However, the use of adenovirus as a vector is presently preferred.

The viral vector of the present invention has a function including a foreign nucleic acid. Delivery of the heterologous nucleic acid facilitates replication of the heterologous nucleic acid in the target cell, followed by expression of the heterologous protein therein. Heterologous protein is defined herein as a protein or a 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 viral vector (eg, the wild-type adenovirus genome) used to deliver the gene to the cell. The term “nucleic acid sequence” or “gene sequence” as used herein shall mean a nucleic acid molecule (preferably DNA). Such gene sequences can be obtained from various sources, including DNA, cDNA, synthetic DNA, RNA or combinations thereof. Such a gene sequence may include genomic DNA, which may or may not contain natural introns. In addition, such genomic DNA can be obtained with a promoter sequence or poly-adenylation sequence. The gene sequence of the present invention is preferably a cDNA. The genome or cDNA can be obtained in a number of ways. Genomic DNA can be extracted and purified from appropriate cells by means known in the art. Alternatively, mRNA can be isolated from the cells and used to prepare cDNA by a reverse transcription reaction or other means.

[0015] Standard techniques for making the vectors of the present invention are known to those of skill in the art, and are described in Sam.
Brook et al., Molecular Cloning: Experimental Manual, Second Edition (Cold Spring Harbor, NY, 1989). A variety of strategies are available for ligating DNA fragments, the choice of which depends on the nature of the ends of the DNA fragment, which can be easily made by one skilled in the art.

As will be appreciated by those skilled in the art, the nucleotide sequence of the inserted heterologous gene sequence or sequences can be any nucleotide sequence. For example, the inserted heterologous gene sequence may (or may not) include a receptor gene sequence or a selectable marker gene sequence. As used herein, a reporter gene sequence is any gene sequence that, upon expression, results in the production of a protein whose presence or activity can be monitored. Examples of suitable reporter genes include genes for galactokinase, β-galactosidase, chloramphenicol acetyltransferase, β-lactamase, and the like. Alternatively, the reporter gene sequence may be any gene sequence whose expression product produces a gene product that affects cell physiology.

[0017] A selectable marker gene sequence is any gene sequence whose presence allows the expression of a protein that allows for the selective growth of cells containing it. Examples of selectable marker genes are those that can confer host resistance to an antibiotic (eg, puromycin, ampicillin, tetracycline, kanamycin, etc.), or that confer host resistance to amino acid analogs, or that lack an additional carbon source. Contains gene sequences that allow the growth of bacteria under possible culture conditions. The gene sequence is
It may be both a reporter gene and a selectable marker gene sequence. The most preferred reporter gene of the present invention is E. coli. A lacZ gene encoding β-galactosidase activity of E. coli; and a gene encoding puromycin resistance.

Preferred reporter or selectable marker gene sequences are sufficient to allow recognition or selection of the vector in normal cells. In one embodiment of the present invention,
The reporter gene sequence encodes an enzyme or other protein whose presence is not normally present in mammalian cells, and whose presence can clearly establish the presence of the vector in such cells.

The heterologous gene sequence may also include a coding sequence for a desired product, such as a suitable bioactive protein or polypeptide, an immunogenic or antigenic protein or polypeptide, or a therapeutically active protein or polypeptide. Preferably, the heterologous gene sequence may encode a therapeutically active protein or polypeptide. In one particularly preferred embodiment, the heterologous gene sequence encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active analog, fragment, or derivative thereof. Alternatively, a heterologous gene sequence can include a sequence that is complementary to an RNA sequence, such as an antisense RNA sequence, which, when administered to an individual, can inhibit expression of the complementary polynucleotide in cells of the individual.

[0020] Expression of the heterologous gene can provide an immunogenic or antigenic protein or polypeptide to achieve an antibody response, which can be collected from a body fluid such as blood, serum or ascites of the animal.

As the inserted heterologous gene sequence, it is also possible to use a gene sequence which already has a promoter, a starting sequence or a processing sequence.

B. Non-viral vector In another preferred embodiment, the transfer vector is non-viral. Other suitable vectors are oligonucleotides (including RNA, DNA, synthetic and modified nucleic acids), plasmids, and E. coli. RNA / DN described by Kometz
A, including but not limited to chimeric molecules. Oligonucleotide vectors include antisense oligonucleotides and oligonucleotides that function as ribozymes. A non-viral transfer vector of the invention can include a foreign nucleic acid as described herein above for the viral vector. Oligonucleotides, plasmids, and RNA / DNA chimeric molecules can be synthesized or produced by any suitable method known in the art.

C. 7 transmembrane receptor ( 7-TM receptor) The receptor that can be used in the practice of the present invention belongs to the 7-TM receptor family. In general, Watson et al., G-protein coupled receptor (G-Pro
tein Linked Receptor, Academic Press, New York (1994); King et al., U.S. Patent No. 5,482,835.

[0024] One skilled in the art will appreciate that the 7-TM receptor is a G protein-coupled receptor. Any mammalian G protein-coupled receptors, and nucleic acid sequences encoding these receptors, can be used in the practice of the present invention. Examples of such receptors include dopamine receptors, muscarinic cholinergic receptors, α
-Including but not limited to adrenergic receptors, opiate receptors, cannabinoid receptors, serotonin receptors, β-adrenergic receptors and purine receptors. As used herein, the term "receptor"
It is intended that the subtype of the named receptor, and variants and homologs thereof, be included, along with the nucleic acid sequence encoding them.

Preferably, the 7-TM receptors used in accordance with the present invention are purine receptors (eg, P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ and P2Y ₁₁ ), adenosine receptors (ie, A1 , A2 and A3,
And its subtypes), bradykinin receptors (e.g., a BK _I and BK _{I I),} or β- adrenergic receptors (e.g., beta _1, beta ₂ and beta _{3). C5A} complement receptors are also preferred. More preferred are P2Y ₂ , BK _II , A _2B
, Β ₂ , and C _5A receptors, with the P2Y ₂ receptor being most preferred. Thus, ligands that can be used in the practice of the invention include nucleotides, nucleosides, catecholamines (eg, dopamine, 5-hydroxytryptophan), C5A
, And bradykinin (s).

P2Y ₂ (also known as P _2U ) -purine receptor undergoes internalization upon activation by ATP, UTP and its analogs. These receptor types are abundant on the luminal surface of human respiratory epithelium. Mason, S.M. J. Et al., 1991
, Br. J. Pharmacol. 103, 1649-1656. P2 of AdV
Activation of these receptors by molecular conjugates to Y ₂ -receptors and then by ATP / UTP causes the vector-ligand-receptor complex to internalize into endosomes and thus to adV into well-differentiated (WD) epithelium. An alternative route of entry is provided, after which the gene is expressed.

D. Antibodies As shown in FIGS. 1 and 2, one strategy for targeting transfer vectors with heterologous nucleic acids to the 7-TM receptor for internalization into cells is to use bispecific cross-linked antibodies. Generally, bispecific antibodies are directed against both the transfer vector and the 7-TM receptor epitope of interest (ie, a binding region that specifically recognizes the transfer vector and a binding region that specifically recognizes the 7-TM receptor). ), Thus forming a "bridge" between the transfer vector and the receptor. Binding of the bispecific cross-linking antibody to the 7-TM receptor induces receptor internalization. The bound antibody-transfer vector complex is internalized with the 7-TM receptor, and thus the transfer vector with the heterologous nucleic acid is introduced 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 adenovirus fiber protein. .

The term “antibody” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Among these,
IgM and IgG are particularly preferred. Antibodies can be monoclonal or polyclonal, can be of any species, including (for example) mouse, rat, rabbit, horse, or human, or can be chimeric antibodies. For example, M. Walker et al., Molec. Immunol. 26, 403-11 (1989). The antibody may be a recombinant monoclonal antibody produced according to the methods disclosed in written US Pat. No. 4,474,893, or in Cabilly et al., US Pat. No. 4,816,567. Antibodies may be made chemically with specific antibodies according to the methods disclosed in SegAl et al., US Patent No. 4,676,980.

The antibodies may be polyclonal or monoclonal, preferably monoclonal. In certain embodiments, the antibodies are cross-linked antibodies specific for both the target receptor and the transfer vector. In this embodiment, the cross-linked antibody is
Preferably, it is a monoclonal antibody directed against the adenovirus fiber (nodule) protein. Also preferred are monoclonal and cross-linked antibodies, including monoclonal antibodies directed against specific epitopes of the 7-TM receptor of interest.

[0030] Antibodies that bind to the same epitope (ie, specific binding site) bound to the 7-TM receptor by the antibody are known in the art, including their ability to compete with the labeled antibody for the 7-TM receptor in a competitive binding assay. Can be identified according to technology.

Antibody fragments within the scope of the invention include, for example, Fab, F (ab ′) 2, and Fc fragments, and corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques.

[0032] Polyclonal antibodies used in the practice of the present invention can be obtained from suitable animals (eg, rabbits,
Goats) can be produced by immunizing with an antigen, to which a monoclonal antibody against the 7-TM receptor binds, collecting immune sera from the animals, and separating polyclonal antibodies from the immune sera according to known procedures. .

[0033] Monoclonal antibodies used in the practice of the present invention include Kohler and Mils.
tein, Nature 265, 495-97 (1975).
It can be produced in a hybridoma cell line. For example, a solution containing the appropriate antigen is injected into a mouse, and after a sufficient time, the mouse is sacrificed to obtain splenocytes. The splenocytes are then immortalized by fusing them with myeloma or lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
The hybridoma cells are then grown in a suitable medium, and the supernatant is screened for monoclonal antibodies with the desired specificity. Monoclonal F
Ab fragments can be produced in Escherichia coli by recombinant techniques known to those skilled in the art. For example, W.S. Huse, Science 246, 1275-81 (198
9).

[0034] Antibodies specific for the 7-TM (eg, P2Y ₂ ) receptor can also be obtained by phage display known in the art.

Those of skill in the art are familiar with many specific immunoassay formats and variations thereof that may be useful in practicing the methods disclosed herein. Generally, E.I. Maggio
, Enzyme Immunoassay, (1980) (CRC Press, Boca Ratone, FL)
See also Skold et al., U.S. Pat. No. 4,727,022, entitled "Methods of Modulating Ligand-Receptor Interactions and Applications", Forrest et al., U.S. Pat. No. 4,659,678, entitled " Immunoassay ", David et al., U.S. Pat. No. 4,376,110, entitled" Immunoassay using monoclonal antibodies ", Litman et al., U.S. Pat. No. 4,275,149, entitled" Macromolecules in Specific Receptor Assays " Environmental Control ", Maggio et al., U.S. Pat. No. 4,233,402, entitled" Reagents and Methods Using Channeling ", Bo.
See US Pat. No. 4,230,767 to Guslaski et al., entitled "Heterospecific Binding Assay Using Coenzymes as Labels." Applicants specifically incorporate the disclosures of all U.S. patents cited herein by reference.

The antibodies described herein can be used according to known techniques, such as precipitation, on a solid support suitable for diagnostic assays (eg, beads, plates, slides or wells formed from materials such as latex or polystyrene). Can be conjugated. Antibodies described herein, according to the known art, radiolabeled (e.g. ^{35 S, 125 I, 1 31} I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (such as fluorescein), such as The detection group can be similarly conjugated. As used herein, "antigenic equivalent" means a protein or peptide that binds to an antibody that binds to a protein or peptide for which equivalence is sought. The antibodies used to select for such antigenic equivalents are referred to herein as "selected antibodies."

E. Non-Antibody-Based Targeting Strategy The invention has been described above with respect to bispecific cross-linked antibodies as a means of targeting transfer vectors to 7-TM for internalization. As shown in FIG. 2, alternative targeting strategies include those using peptides and 7-TM receptor agonists (agonists) / antagonists (antagonists).

For peptides, the peptide can be a natural ligand that binds to the 7-TM receptor. Peptide agonists and antagonists of the 7-TM receptor are known in the art. In addition, novel 7-TM receptor agonists / antagonists can be identified as described in King et al., US Pat. No. 5,482,835. Alternatively, peptides can be identified as binding to the 7-TM receptor by phage display technology, or any other method in the art.

[0039] Methods for synthesizing or producing peptides are known in the art. In one particular embodiment, the fusion of the nucleic acid encoding the peptide to the gene encoding the AdVknob protein results in the expression of a knob-peptide chimeric protein. For example, it is known that exogenous nucleic acids can be expressed at the C-terminal or H1 loop region of the knob protein. In another embodiment, a concatamer of the peptide is expressed on the knob protein.

As a further alternative, targeting can be achieved using peptides incorporated into the “receptor body”. Generally, the receptor body is a truncated truncated receptor in which the peptide that binds to the 7-TM receptor has been replaced by the intramembrane and intracellular regions of the adenovirus receptor. This means that the outer part of the viral receptor acts as an "antibody" against the binding region of the virus (eliminating the need for an antibody against AdV). For example, the ectodomain of the Adv receptor can be fused to the peptide ligand of the 7-TM receptor.
This complex binds to recombinant AdV and thus displays the bradykinin peptide on the surface of the virus. The peptide ligand fused to the truncated AdV receptor then targets the complex to a cognate receptor for the peptide ligand on the cell surface.

As a still further alternative, the transfer vector can be targeted to the 7-TM receptor by a chemically linked high affinity agonist / antagonist of the 7-TM receptor. The high affinity agonist / antagonist can be a peptide ligand as described above, or can be a nucleotide (eg, A
TP, UTP), dinucleotides (described in more detail herein below), and derivatives thereof. Further, P2Y, which can be used in the practice of the present invention,
Receptor ligands, particularly P2Y ₂ receptor ligands, all compounds are P2Y2 ligands include in particular nucleotides and dinucleotides, W. Pen
Dergast et al., U.S. Pat. No. 5,837,861 (November 17, 1998), to Jacobs and Leighton, U.S. Pat. No. 5,763,447.
U.S. Pat. No. 5,789,391 to Javabus et al., U.S. Pat. No. 5,635,160 to Stutss et al., And U.S. Pat. No. 5,29 to Boucher et al.
No. 2,498, the disclosure of which is incorporated herein by reference in its entirety.
The specification is incorporated herein by reference.

Examples of such nucleotides are shown in formulas I-IV. Formula I is

Embedded image , And the formula, X _1, X ₂ and X ₃ are each independently, O ^- or S ^- and is, preferably, X ₂ and X ₃ are O ^- a and, R ₁ is, O, imido , Methylene or dihalomethylene (eg, dichloromethylene or difluoromethylene), preferably, R ₁ is either oxygen or difluoromethylene, and R ₂ is either H or Br, preferably , R ₂ is H and the formula I
Particularly preferred compounds of uridine 5′-triphosphate (UTP) and uridine 5
'-O- (3-thiotriphosphate) (UTPγS).

A dinucleotide is a compound represented by the general formula II. Formula II is

Embedded image Wherein X is any of oxygen, methylene, difluoromethylene, imide, n = 0, 1, or 2, and m = 0, 1, or 2. , N + m = 0, 1, 2, 3, or 4, and B and B
'Are each independently either purine residue or a pyrimidine residue linked through the respective 9 or 1-position, a Z = OH or N _3, Z' = OH
Or N ₃ , Y = H or OH, and 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 β configuration. The furanose sugar is most preferably in the β-D configuration.

Preferred compounds of the formula II are those of the formula IIa Formula IIa is

Embedded image Where X = O, n + m = 1 or 2, Z, Z ′, Y and Y ′ = OH, and B and B ′ are defined by formulas IIc and IId. X = O, n + m = 3 or 4, Z, Z ′, Y and Y ′ = OH, B = uracil, B ′ is defined by formulas IIc and IId.
Or X = O, n + m = 1 or 2, Z, Y, and Y ′ = OH, Z ′ = H, B = uracil, and B ′ is defined by formulas IIc and IId Is done. Or X = O, n + m = 0, either 1 or 2, Z and Y = OH, Z ′ = N ₃ , Y ′ = H, B = uracil, B '= Thymine. X = O, n + m = 0, either 1 or 2, Z and Z ′ = N ₃ , Y and Y ′ = H, and B and B ′ = thymine. Or X is any of CH ₂ , CF ₂ , or NH;
n and m = 1, Z, Z ′, Y and Y ′ = OH, and B and B ′ are defined by formulas IIc and IId.

Another preferred group of compounds of formula II is a compound of formula IIb or a pharmaceutically acceptable salt thereof. Formula IIb is

Embedded image It is. Wherein X is any of oxygen, methylene, difluoromethylene, or imide, n = 0 or 1, m = 0 or 1, and n + m = 0, 1
, Or 2; B and B ′ are each independently 9
A purine residue, such as formula IIc, linked through position or position 1, or a formula IId
And a pyrimidine residue. When B and B 'are uracils attached to the ribosyl moiety at position N-1, m
The sum of + n can be equal to 3 or 4 when X is oxygen. The ribosyl moiety is in the D configuration, as shown, but may be in the L configuration, or in the D and L configurations. The D configuration is preferred. Formula IIc is

Embedded image It is.

The substituted derivative of adenine is adenine 1-oxide, 1, N6- (4- or 5
R- of 6- or 8-HNR 'comprises arylalkyl (-substituted etheno) adenine, 6-substituted adenine, or 8-substituted aminoadenine, wherein the aryl moiety is optionally functionalized as described below. a C ₁ ~ ₆₎ group, and alkyl, for example ([6
-Aminohexyl] carbamoylmethyl)-, and ω-acylated-amino (hydroxy, thiol and carboxy) derivatives wherein the acyl group is derived from, but not limited to, acetyl, trifluoroacetyl, benzoyl, substituted benzoyl and the like. Or the carboxyl moiety is selected from an alkyl group having a functional group such as an ester or amide derivative thereof, such as an ethyl or methyl ester or a methyl, ethyl or benzamide derivative thereof. ω- amino (hydroxy, thiol) moiety may be alkylated with C ₁ ~ ₄ alkyl group.

Similarly, B or B ′ or both of formula IIb are linked via position 1.
It can be a pyrimidine having the general formula of formula IId. Formula IId is

Embedded image It is. Wherein, R ₄ is hydroxy, mercapto, amino, cyano, aralkoxy, is any one of C ₁ ~ ₆ alkoxy, C ₁ ~ ₆ alkylamino, and dialkylamino, the alkyl group is linked optionally form a heterocyclic ring, R ₅ is hydrogen, acyl, is either a C ₁ ~ ₆ alkyl, aroyl, C ₁ ~ ₅ alkanoyl, benzoyl or sulfonate,, R ₆ is hydroxy, mercapto, alkoxy, aralkoxy, C ₁ ~ ₆ - alkylthio is either C ₁ ~ ₅ disubstituted amino, triazolyl, alkylamino or dialkylamino, the alkyl group, linked optionally, either to form a heterocyclic ring, or N -3 to form an optionally substituted ring, ureido or substituted ureide, wherein the pyrimidine ring is separated from Ureido N atom, C ₁ ~ ₆ alkyl group or C ₆ ~ _10,
Substituted by any of the aryl group or C ₇ ~ ₁₂ arylalkyl group, wherein alkyl, aryl and arylalkyl moieties, any amino, hydroxy, thio, carbonyl or sulfonyl substituents (which itself may be substituted) Can be replaced by

R₇Is any of hydrogen, hydroxy, cyano, nitro, and alkenyl;
The alkenyl moiety is optionally linked through an oxygen atom to form a carbon adjacent to oxygen.
Optionally with an alkyl or aryl group, substituted alkynyl or hydrogen
Forming a substituted ring, R₈Is amino or substituted amino and halogen, al
Killed, substituted alkyl, perhalomethyl (eg CF_Three), C_Two~₆Alkyl, C_Two~ _Three Alkenyl or substituted ethenyl (eg, alkylamino, bromovinyl and
And ethyl propenoate or propenoic acid), C_Two~_ThreeAlkynyl or substituted alkynyl
Any of quinyl, R₆Is any other than amino or substituted amino
, R_Five-R₆Together, R₆5 or 6 members linked via N or O at
When a member can form a saturated or unsaturated ring, such ring may itself contain a functional group.
And substituents.

R ₈ is hydrogen, alkoxy, arylalkoxy, alkylthio, arylalkylthio, carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, or phenylthio.

In the general structure of FIG. IId above, the dashed lines at positions 2 to 6 shall indicate the presence of a single or double bond at these positions; Determined by whether ₄ , R ₅ and R ₇ are capable of keto-enol tautomerism.

In the general structure of FIGS. IIc and IId above, the acyl group advantageously comprises an alkanoyl or aroyl group. Alkyl groups are advantageously 1 such as
1 to 8 carbon atoms, especially 1, optionally substituted by one or more suitable substituents
It contains up to 4 carbon atoms. As aryloxy, the aryl group containing the aryl portion of such a group is preferably a phenol group optionally substituted with one or more suitable substituents as described below. The alkenyl and alkynyl groups described above are advantageously optionally substituted with one or more suitable substituents as described below.
It contains 2 to 8 carbon atoms, especially 2 to 6 carbon atoms, such as ethenyl or ethynyl. The above alkyl, alkenyl, alkynyl, and suitable substituents on the aryl groups are advantageously selected from halogen, hydroxy, C ₁ ~ ₄ alkoxy, C ₁ ~ ₄ alkyl,
C ₆ ~ ₁₂ aryl alkoxy, carboxy, cyano, nitro, sulfonamide,
Sulfonate is selected from phosphoric acid, sulfonic, amino, and substituted amino, amino here, is replaced by a single or double by C ₁ ~ ₄ alkyl, optionally substituted on the double, alkyl group, Optionally linked to form a heterocycle.

To further clarify the preceding description of Formulas IIc and IId, the description can be simplified as follows. R ₂ is O or nothing, or R ₁ and R ₂ together can form an optionally substituted 5-membered fused imidazole ring; or R 6 of the 6-HNR ₁ group ₁ or 8-HNR ₃ group R ₃ is, (a) aryl aryl moiety is optionally substituted alkyl (C ₁ ~ ₆₎ group, and (b) alkyl, (c
) (And [6-aminohexyl] carbamoylmethyl), (d) .omega.-amino alkyl (C ₂ ~ _10), and (e) .omega.-hydroxyalkyl _{(C 2 ~ 10), (} f) ω- thiol alkyl and (C ₂ ~ _10), and (g) .omega.-carboxyalkyl (C ₂ ~ _10),
(H) (b), ( c), or ω- acylated derivatives of (d) (acyl groups here, acetyl, trifluoroacetyl, or benzoyl or substituted benzoyl alkyl, (C ₂ ~ ₁₀₎ And (i) ω as in (e) above.
- carboxyalkyl (C ₂ ~ ₁₀₎ (carboxyl moiety here is a is an ester or amide) selected from the group consisting of a.

Formula IId is

Embedded image It is. Wherein, R ₄ is hydroxy, mercapto, amino, cyano, aralkoxy, is any one of C ₁ ~ ₆ alkylthio, C ₁ ~ ₆ alkoxy, C ₁ ~ ₆ alkylamino or dialkylamino, wherein the dialkylamino here alkyl groups may form a heterocyclic ring linked optionally, R ₅ is hydrogen, acyl, C ₁ ~ ₆ alkyl, aroyl, is any one of C ₁ ~ ₆ alkanoyl, benzoyl or sulfonate, R ₆ is hydroxy, mercapto, alkoxy, aralkoxy, is any one of C ₁ ~ ₆ alkylthio, C ₁ ~ ₅ disubstituted amino, triazolyl, alkylamino or dialkylamino, alkyl groups of said dialkylamino here, linked optionally, either to form a heterocyclic ring, or linked to N ^3,
R ₅ -R ₆ together form a 5- or 6-membered saturated or unsaturated ring linked via N or O at R ₆ , wherein the ring is optionally substituted. R ₇ is (a) hydrogen, (b) hydroxy, (c)
Cyano, (d) nitro, and (e) alkenyl, wherein the alkenyl moiety is optionally linked via oxygen and optionally substituted with an alkyl or aryl group on a carbon adjacent to oxygen. And (f) substituted alkynyl;
g) halogen, and (h) alkyl, and (i) substituted alkyl, and (j) perhalomethyl, and (k) C ₂ ~ ₆ alkyl, and (l) C ₂ ~ ₃ alkenyl, and (m) substituted ethenyl , (n) and C ₂ ~ ₃ alkynyl and is selected from the group consisting of a (o) substituted alkynyl (if R ₆ is other than amino or substituted amino), R ₈ is, (a) hydrogen, (b) (G) alkoxy, (c) arylalkoxy, (d) alkylthio, (e) arylalkylthio, (f) carboxamidoethyl,
Selected from the group consisting of carboxymethyl, (h) methoxy, (i) methylthio, (j) phenoxy, and (k) phenylthio.

CTP and its analogs have the general formula III:

Embedded image Wherein R ₁ , X ₁ , X ₂ and X ₃ are defined by formula I and R ₅ and R ₆ are H
While R ₇ is nothing and there is a double bond between N-3 and C-4 (cytosine) or R ₅ , R ₆ and R ₇ together are —CH ＝ CH— Oh, N-
4 and N-3 with a double bond between C-3 to N-4 of the ring is formed and is optionally substituted at the 4-position or 5-position of the etheno ring (3, N ⁴ - Etenocytosine)].

ATP and its analogs have the formula IV:

Embedded image Wherein R ₁ , X ₁ , X ₂ , and X ₃ are defined by Formula I, wherein R ₃ and R ₄ are H, while R ₂ is absent and N-1 and C-6 Whether there is a double bond between
Adenine), or R ₃ and R ₄ are H, while R ₂ is O, N-1 and C
-6, there is a double bond (adenine 1-oxide), or R ₃ , R ₄ , and R ₂ together are —CH ＝ CH—, and between N-6 and C-6 (1, N6-ethenoadenine) having a double bond to form a ring from N-6 to N-1].

For simplicity, the formulas I, II, III and IV herein refer to active compounds in the natural D configuration, but the invention also encompasses, unless otherwise specified, compounds in the L configuration, and Also included are mixtures of compounds in the D and L configurations. The natural D configuration is preferred.

Some compounds of formulas I, II, III and IV can be prepared by methods known to those skilled in the art and according to known procedures (Zamecnik, P et al., Proc
. Natl. Acad. Sci. USA 89: 2370-2373 (1992)
); Ng, K .; Et al., Nucleic Acids Res. 15: 3572-3
580 (1977); Jacobs, K .; M. Et al., US Pat. No. 5,789,3.
No. 91 and Pendergast, W.C. Et al., International Patent Application No. WO 98/349.
No. 42); some are described, for example, in Sigma Chemical Co., PO Box 14508,
Commercially available from St. Louis, MO 63178. U.S. Pat. No. 5,789,
No. 391 and International Patent Application WO 98/34942 are hereby incorporated by reference.

In yet another embodiment, a high affinity agonist or antagonist can be directly linked to a transfer vector using sulfo-N-hydroxysuccinimide (NHS), as described in more detail below. .

F. Examples of compounds that can be used in the practice of the present invention include biotin and covalent conjugates include compounds of formulas I-IV above,
General formula:

Embedded image Wherein X can be O or S, and A is a purine or pyrimidine base (eg, adenine, guanine, thymine, cytosine, uracil) (each purine or pyrimidine base is preferably a ribose or deoxyribose ring In the case of purines, they are connected by a covalent bond to 9 nitrogens, in the case of pyrimidines they are connected by a covalent bond to 1 nitrogen, R ₁ is either H or OH, and n is 1-4 or 6
And preferably in any of the 2-, 3-, or 4-positions].

The transfer vector may be connected or conjugated covalently or non-covalently to a purine or pyrimidine base (eg of a compound of formulas I-IV above) or to the corresponding ribose or deoxyribose ring,
Alternatively, the terminal phosphate moiety of the compounds represented by Formulas I, II and III above may be attached to the terminal phosphate moiety by any suitable means, for example, at any appropriate position (eg, carbon 5 at pyrimidine or carbon 2 at purine). , Ring carbon such as 6-position carbon or 8-position carbon)
To a linking group to which a ligand can be covalently attached, or to a linking group to which a biotin group can be attached, wherein the biotin group is covalently attached to the ligand (see below). Connected, and the two biotin groups are attached by connecting via a covalent bond, wherein both biotin groups are connected to each other by a connected or conjugated avidin group).

Specific examples of ligands that can be used in the practice of the present invention have the general formula:

Embedded image Wherein X can be O or S, and A and B are each independently a purine or pyrimidine base (eg, adenine, guanine, thymine, cytosine, uracil), preferably A or B Is uracil, and in one particular preferred embodiment, A is uracil, B is cytosine, and R ₁ and R ₂ are each independently selected from the group consisting of H or OH; n is 1 to
4 or 6, preferably 2, 3 or 4, and the transfer vector is directly linked to A or B or to the ribose or deoxyribose ring to which A or B is connected. Or indirectly, conjugated or connected by a linking group, covalently or non-covalently, in the same manner as described above.

[0062] In one particular embodiment, biotin (B) -UTP is used as a targeting agonists of P2Y ₂ receptors. B-UTP interacts with a biotinylated virus transfer vector in the presence of streptavidin (SA) to give a virus-biotin-SA-UTP complex, which is P2Y ₂
Target to the receptor. Alternatively, oligonucleotides, plasmids and RNA / DNA chimeric molecules can be synthesized or produced to incorporate B-UTP or any other suitable labeled nucleotide.

A specific example of a biotin-dinucleotide conjugate is of the formula:

Embedded image Wherein the biotin moiety is linked to the pyrimidine base by an aminoallyl linker; another specific example of a biotin-dinucleotide conjugate is of the formula:

Embedded image Wherein the biotin moiety is linked to the pyrimidine base by a linker attached by alkylation of the thiol group.

Alternatively, a P2Y ligand conjugate can be formed by linking a biotin moiety or by linking directly to a vector via an etheno moiety fused to a purine or pyrimidine base.

Particular examples of etheno-linked conjugates include the general formula of optionally substituted ethenocytidine and ethenoadenosine triphosphate and analogs thereof:

Embedded image

Embedded image Wherein the linker is any linear or aromatic (e.g., phenyl, naphthyl) of 2 to 24 atoms in length, optionally substituted and covalently linking the etheno moiety to the functional moiety, or Indicates a heterocycle (e.g., benzothiophene, isoxazole, pyridine, piperidine) and the function is any of the methods described herein useful for introducing heterologous nucleic acids into cells by internalization of a receptor-ligand complex. And R ₁ and R ₂ can be either H or OH, X is any of CH ₂ , CCl ₂ , CF ₂ , NH or O, and Y is O or S] It is a structure that is

Other preferred embodiments of the etheno-linked conjugate are any of the ethenocytidine and ethenoadenosine dinucleotide compounds of the formula: wherein the linker is attached at position 4 or 5 of the etheno moiety. obtain.

Embedded image Wherein the linker is any linear or aromatic (e.g., phenyl, naphthyl) of from 2 to 24 atoms in length, optionally substituted and covalently linking the etheno moiety to the functional moiety, or Indicates a heterocycle (e.g., benzothiophene, isoxazole, pyridine, piperidine) and the function is any of the methods described herein useful for introducing heterologous nucleic acids into cells by internalization of a receptor-ligand complex. Where R ₁ and R ₂ can be H or OH.

Additional preferred compounds are 6- and 8-substituted adenosine triphosphates and dinucleotides thereof, as described in Formula II.

The linking of the heterocyclic base to the carbon can be accomplished by using a photoactivatable linker, such as, but not limited to, using a commercially available biotin derivative (Pierce Biochemicals, Immunopure Photoactivatable Biotin). Can be created via

Alternatively, P2 nucleotide ligand conjugates can be attached by attaching a biotinyl moiety via a linker, or by derivatization (eg, acylation, alkylation) of a sugar hydroxyl group, or to a compound of formulas I-IV above. It may be formed by linking the vector directly to the sugar moiety of the nucleotide via a heteroatom surrogate (eg, SH, NHR) of the hydroxyl group of the molecule shown.

The P2 nucleotide ligand conjugate can also be attached to the biotinyl moiety via a linker, or directly to the terminal phosphate moiety of the molecule represented by Formulas I, III and IV above via a linker. Can be formed.

The linking groups used in the practice of the present invention are generally polymers that include both water-soluble and water-insoluble polymers. Water-soluble or hydrophilic linking groups are preferred. A polymer is an elongated, flexible chain of repeating monomer units, which may or may have functional groups along its length. Numerous polymers that are functionalizable, typically covalently, to function as linking groups for ligands and vectors are known and will be readily apparent to those skilled in the art. Examples include, but are not limited to, polysaccharides such as dextran, polypeptides such as polyvinyl alcohol, polylysine, and polyacrylic acid. Ligands and vectors can be linked to a linking group (including its free chain end) in any conformation or position. Generally, a linking group comprises a chain of 2 to 24 atoms, such as carbon atoms, optionally substituted as described above.

Biotin is covalently connected to the ligand by conventional techniques, and both biotin groups can be connected to avidin or streptavidin groups according to known techniques to form a conjugate of vector and ligand.

Examples of ligands to which biotin is connected by a covalent bond are:

Embedded image Wherein R ₂ is H or OH, preferably OH, and n is any of 1-4, preferably equal to either 2 or 3. Such compounds are known and are commercially available. The uracil group shown can be substituted with either another purine or pyrimidine base as described above, and the biotin and the connecting polymer chain shown between the biotin group and the uridine group can be substituted at any suitable position (eg, , 5 carbons of a pyrimidine, or a ring carbon such as 2 or 8 carbons of a purine). Importantly, oligonucleotides (eg, DNA, RNA or chimeras of 5 or 10 to 30 or 50 bases) can be synthesized using one or more bases thus conjugated to biotin, Biotinylated oligonucleotides can be conjugated to a biotinylated ligand as described herein using avidin.

The biotin group can be added to a vector (particularly a vector having a free amine group such as a viral vector) by Pierce (3747N. Meridian, PO Box.
117, Rockford, IL 61105) and can be connected covalently using the EZ-LINK® sulfo-NHS-LC-biotinylation kit. Examples of compounds that can be used for biotinylation of a primary amine on a vector include:
Available from earrings, structure:

Embedded image With sulfo-NHS-LC-biotin.

In another embodiment, removing the biotin group shown in the sulfo compound above and replacing it with a covalent bond to a ligand as described above can provide a direct covalent linkage from the vector to the ligand. .

G. Target cells Hematopoietic stem cells, lymphocytes, vascular endothelial cells, respiratory epithelial cells, keratinocytes, skeletal muscle and cardiomyocytes, nerves and cancer cells are proposed targets for therapeutic gene transfer in vitro or in vivo include. For example, A. D. Miller, N
atature 357, 455-460 (1992); C. Mulligan
, Science 260, 926-932 (1993). These 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 normally bind to transfer vectors, ie, viral vectors.

In particular, any cell that expresses a receptor from the 7-TM receptor family is a suitable target for use in the present invention. Preferred are purine receptors (eg, P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ , P2Y ₁₁ ), adenosine receptors (ie, A1, A2, A3), bradykinin receptors (eg, BK _I , BK _II ), β Adrenergic receptors (eg, β ₁ , β ₂ , β ₃ )
Or a cell that expresses the _C5A complement receptor. More preferred is P2Y ₂
, BK _II , A _2B , β ₂ , C _5A receptors, and most preferably P2Y ₂ receptor. Also preferred as targets are respiratory epithelial cells, especially differentiated epithelial cells of the cylindrical airways. Cells can be administered with the conjugate in vitro or in vivo, for example, by administering an aerosol containing the conjugate to the luminal surface of epithelial cells of the respiratory tract.

Thus, purine receptors that can be used in accordance with the present invention include P2 purine receptors. Numerous P2 purine receptors are known. For example, P2 purine receptors: local, functional and transduction mechanisms, Chadwick and J.W. G
oed, 1996. P2 purine receptors include P2X, P2Y, P2T
Includes P2U (now called P2Y ₂₎ and P2Z purine receptors (including its subclasses). More recently, purine receptors have been classified as a P2Y family consisting of G protein-coupled receptors and a P2X family consisting of ligand-gated cation channels. P2Z purine receptors that open non-selective pores can be considered a third family. Ibid 6-7. As different purine receptors are found in different tissues, the transforming tissue or group of tissues can be determined, in part, by the choice of targeting purine receptor, and the corresponding ligand can be targeted, as discussed in more detail below. Selected purine receptors. Of course, it is understood that many ligands bind to multiple receptor subtypes.

The P2X ₁ receptor can be used for targeting vas deferens (eg, for the production of immunocontraceptive vaccines). The ligands that can be used for this receptor are 2
-Methylthio ATP (2-MeSATP), ATP, and α, β-methylene A
Including, but not limited to, TP (α, β-meATP).

[0080] Immune system cells, including but not limited to monocytes, macrophages, mast cells, neutrophils and B cells, can be targeted using P2Y and / or P2U receptor ligands, as well as P2Z receptor ligands. Such cells are:
The host subject can be transformed with a nucleic acid that expresses an immunogen that is effective to generate an immune response against the antigen or disease vector.

[0081] Pancreatic cells, such as pancreatic B cells, can be targeted with a P2Y receptor ligand. Such cells can be transformed with a nucleic acid that expresses insulin in the cells in an amount effective to address the subject's hypoglycemia or diabetes.

The P2X ₃ , P2X ₄ , P2X ₅ , and P2X ₆ receptors can be used for targeting neural tissue. Ligands that can be used for these receptors include 2-M
eSATP, ATP, and (for P2X3 and P2X4) α, β-meAT
Including but not limited to P.

The P2Z receptor can be used for macrophage targeting. Ligands that can be used for these receptors include, but are not limited to, 3'-O- (4-benzoyl) benzoyl ATP (BzATP), ATP, and UTP.

The P2Y ₁ receptor can be used for targeting neural, placental and endothelial tissues. Ligands that can be used for this receptor include 2-MeSATP, AT
Including but not limited to P, ADP, and UTP.

The P2Y ₂ receptor may be used for targeting lung, bone, and pituitary tissue. Ligands include, but are not limited to, ATP, UTP, and 2-MeSATP.

[0086] P2Y ₃ receptors, may be used in the targeting of nerve tissue. Ligands include but are not limited to ADP, UTP, ATP and UDP.

The P2Y ₄ receptor can be used for targeting of placenta and nerve tissue. Ligands include, but are not limited to, UTP, UDP, ATP and ADP.

[0088] P2Y ₅ receptor, it may be used in the targeting of lymphocytes. Ligands include but are not limited to ADP, ATP and UTP.

The P2Y ₆ receptor may be used for targeting blood vessels, especially smooth and arterial smooth muscle tissue. Ligands include UTP, ADP, 2-MeSATP and A
Including but not limited to TP.

Additional examples of P2 purine receptor ligands (and the corresponding receptor subtypes to which they bind) that may be used in the practice of the invention include P2 purine receptor antagonists, such as: quinidine (P2X and P2Y receptors); phentolamine Imidazoline (P2Y receptor); 2,2′-pyridylisatogen tosylate, ie, “PIT” (P2Y receptor); 3-O-3 [N- (
4-azido-2-nitrophenyl) amino] propionyl ATP, ie "A
NAPP3 "(P2X receptor): apamin (P2Y receptor); α, β-
meATP (P2X receptor); Reactive Blue 2 (P2Y, P2X and P2
Z receptor); Suramin (P2X, P2Y, P2T and P2Z receptors)
8-β, 5-dinitrophenylenecarbonylimino) -1,3,5-naphthalene trisulfonate, ie “XAMR0721” (P2Y receptor); pyridoxal phosphate-6-azophenyl-2 ′, 4′-disulfonic acid, "PPADS" (P2X and P2Y receptors); pyridoxal phosphate-6
-Azophenyl-2 ', 5'disulfonic acid, ie, "isoPPADS" (P2
Pyridoxal-5-phosphate, ie, “P5P” (P2X receptor); 4,4′-diisothiocyanostilbene-2,2′-disulfonate, ie, “DIDS” ((P2X receptor); Evans Blue, trypan blue, and Congo red (P2X receptor); brilliant blue (P2
Z receptor); oxidized ATP, ie, “o-ATP” (P2Z receptor, for targeting macrophages); 2-propylthio-D-β, γ-difluoromethylene ATP, ie, “ARL66096” (for P2T receptor, platelet targeting). Including.

H. Gene Transfer The methods of the present invention provide a means for delivering heterologous nucleic acids independent of the target cell nucleus to a broad phylogenetic range of host cells. The vectors, methods and pharmaceutical formulations of the present invention are further useful for methods of treating or otherwise administering a protein or peptide to a subject in need of the desired protein or peptide. Thus, the protein or peptide can be produced in vivo in a subject. The subject may be deficient in the protein or peptide, or because the production of the protein or peptide in the subject may confer some therapeutic effect, as a treatment or otherwise, as described further below. In addition, the subject may need a protein or peptide.

[0092] The gene transfer engineering of the present invention has several applications. Perhaps the most immediate application is the processing of peptides and the elucidation of functional domains of proteins. To study cell-specific differences in processing and cell fate, cloned cDNA or genomic DNA sequences of the protein can be introduced into various cell types in culture or in vivo. By substituting coding sequences under the control of a strong promoter, substantial amounts of the desired protein can be made. In addition, specific residues involved in protein processing, intracellular localization, or biological activity can be determined by mutation of discrete residues in the coding sequence.

The gene transfer engineering of the present invention can also be applied to control protein expression and evaluate the ability to regulate cellular events. Some functions of the protein, such as its role in differentiation, may be studied in tissue culture but need to be reintroduced into in vivo systems at different stages of development to monitor changes in the associated properties There are also things.

Gene transfer provides a means of studying nucleic acid sequences and cellular factors that regulate the expression of specific genes. One approach to such studies would be to fuse the regulatory element to be studied to a reporter gene and then assay for reporter gene expression.

Gene transfer also has substantial potential use in elucidating and providing treatments for disease states. There are many congenital diseases for 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. A state of deficiency, usually enzymatically inherited, usually of enzymes, and an imbalance in which, at least occasionally, a regulatory or structural protein is involved and inherited dominantly. For deficiency conditions, replacement of the gene can be used to bring the normal gene to the affected tissue using gene transfer, and animal models of the disease can be created using antisense mutations. In imbalanced disease states, gene transfer can be used to create a disease state in a model system, which can then be used in an effort to negate the disease state. Thus, the methods of the present invention allow for the screening of compounds useful for treating genetic diseases, eg, addressing disease states. A disease state, as used herein, causes disease or
Or by partially or completely healing the deficiency or imbalance that exacerbates it. The use of site-specific integration of nucleic acid sequences to cause mutations or correct defects is also possible.

In one particularly preferred embodiment, the present invention is used to express exogenous CFTR protein in respiratory epithelia. According to this embodiment, the use of an AdV transfer vector having the CFTR gene is preferred. AdV-CFTR, as described above, using sulfo--NHS activator, directly linked to 4-amino substituent of the dinucleotide UP ₄ C. Binding of UP ₄ U to P2Y ₂ receptor on the tip surface of the respiratory epithelium, induce onto splenocytes internalization of all UP ₄ C-AdV-CFTR complex.

I. Pharmaceutical Formulations, Subjects, and Methods of Administration Subjects suitable for treatment according to the present invention include both birds and mammals, preferably mammals. Any mammalian subject in need of treatment according to the present invention is suitable, but human subjects are preferred. Human subjects of both genders and at any stage of development (ie, neonate, infant, young, adolescent, adult) can be treated according to the present invention. Preferred is a human subject suffering from cystic fibrosis.

Compounds of the present invention may include an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as manganese, magnesium, or calcium; or an ammonium or tetraalkylammonium salt, ie, NX ₄ ⁺ (where X is C (But not limited to ₁ to ₄ ) in the form of a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.

The active compounds of the present invention can be administered to a subject in need thereof by oral, rectal, transmucosal, topical or enteral administration, intramuscular, subcutaneous, intramedullary injection. Parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection can be administered by any suitable means. Alternatively, the compounds may be administered locally, rather than systemically, in a depot or sustained release formulation. Pulmonary administration is preferred.

The active compounds disclosed herein may be administered to a subject's lungs by any suitable means, but preferably, an aerosol suspension of inhalable particles of the active compound, comprising The subject is inhaled). The respirable particles may be liquid or solid.

Aerosols of liquid particles containing the active compound may be prepared by any suitable means, for example using a pressure driven aerosol nebulizer or an ultrasonic nebulizer. See, for example, U.S. Patent No. 4,501,729. Nebulizers are based on the acceleration of compressed gas, typically air or oxygen, through a narrow venturi
Or a commercially available device that converts a solution or suspension of the active ingredient into a therapeutic aerosol mist by ultrasonic agitation. Formulations suitable for use in a nebulizer comprise the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w / w of the formulation, but preferably up to 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, for example, by the addition of sodium chloride.

Optional additives, if the formulation is not sterile, include, for example, preservatives such as methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffers and surfactants.

Aerosols of solid particles containing the active compound can likewise be prepared using any solid particulate pharmaceutical aerosol maker. An aerosol creator for administering a solid particulate medicament to a subject produces particles that are inhalable as described above and a volume containing a predetermined fixed dose of medicament at a rate suitable for human administration. Produces an aerosol. One exemplary type of solid particulate aerosol maker is a duster.
Formulations suitable for administration by a duster include finely divided powders that can be delivered by a duster or can be sniffed and introduced into the nasal cavity. In this duster, powder (for example,
(A dose thereof effective to carry out the treatments described herein) is contained in a capsule or cartridge, typically made of gelatin or plastic,
Puncture or opening in situ, powder is delivered by air drawn through the device upon inhalation or by a manually operated pump. The powder used for the duster is
Consisting of the active ingredient alone 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 0.1-100% w / w of the formulation. A second type of exemplary aerosol creator includes a fixed dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution of the active ingredient in a liquefied propellant. In use, these devices pump the formulation through a valve suitable for delivering a constant volume, typically 10-150 μl, to generate 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 further comprise one or more co-solvents, for example, ethanol, a surfactant, for example, sorbitan oleate or trioleate, an antioxidant and a suitable flavoring agent.

The aerosol (formed from solid or liquid particles) is an aerosol generator
It can be produced at a rate of about 10-150 liters / minute, more preferably about 30-150 liters / minute, and most preferably about 60 liters / minute. Aerosols containing larger amounts of medicament may be administered more quickly.

The dose of an active compound disclosed herein or a pharmaceutically acceptable salt thereof will vary depending on the condition being treated and the condition of the subject, but will generally vary over the surface of the subject's airways. , About 10 ^-9 or 10 ^-7 to about 10 ^-3 mol / l, more preferably about 1
0 ^-6 can be an amount sufficient to achieve dissolved concentrations of active compound to about 3 × 10 ^-4 mol / liter. The daily dose may be divided into one or several unit dose administrations depending on the solubility of the particular formulation of active compound to be administered. Other compounds may be administered simultaneously with the active compound of the present invention or a salt thereof.

The solid or liquid particulate pharmaceutical formulation comprising the active substance of the present invention should contain particles of inhalable size; that is, particles of a size small enough to reach the bronchi and alveoli through the mouth and larynx during inhalation. It is. Generally, particles in the range of about 1-5 microns in size (more specifically, about 4.7 microns or less) are inhalable. Non-inhalable size particles contained in the aerosol tend to deposit in the throat and be swallowed, and the amount of non-inhalable particles in the aerosol is preferably minimal. For intranasal administration, a particle size in the range of 10-500 μm is preferred to ensure retention in the nasal cavity.

In administering the active compounds of the present invention, they may be administered separately (simultaneously or sequentially).
They may be administered, or alternatively and preferably, they may be premixed and administered as a preformed conjugate. As an illustrative example, an appropriate dose of a transfer vector having a heterologous nucleic acid of interest is premixed with a target molecule (ie, a bispecific cross-linking antibody, a peptide, biotin-UTP, etc.), and the complex is administered to a subject. Can be administered.

In preparing the formulations according to the present invention, the active substance or a physiologically acceptable salt or free base thereof is typically mixed, inter alia, with 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 harmful to the patient. The carrier may be a solid or liquid, or both, and is preferably formulated with the compound as a unit dose formulation, eg, as a capsule, which may contain from 0.5% to 99% by weight of the active compound. 1
One or more active compounds may be incorporated into a formulation of the present invention, which may be prepared by any of the known pharmacological techniques which consist essentially of admixing the components.

A composition comprising inhalable dry particles of the active compound is obtained by milling the active compound using a mortar and pestle and then passing the finely divided composition through a 400 mesh screen,
It can be prepared by breaking or separating large agglomerates.

The pharmaceutical composition may optionally include a dispersing agent that acts to facilitate the formation of an aerosol. A suitable dispersant is lactose, which may be blended with the active in any suitable ratio (eg, a 1 to 1 weight ratio).

In summary, the transfer vectors of the present invention can be used to stably transfect dividing or non-dividing cells and stably express heterologous genes. Using this vector system, it has now become possible to introduce into a dividing or non-dividing cell a gene that encodes a protein that can affect the physiology of the cell. Thus, the vectors of the invention can be used for gene therapy for disease states, or
It may be useful for experimental modifications of cell physiology, such as to create models useful for screening compounds for specific biological activities.

Although the invention has been described above, the same will be described with reference to certain examples, which are included for illustrative purposes only and are not considered limiting of the invention.

[0113]Example 1 Human airway epithelial cell model Gray et al., Am. J. Respir. Cell Mol. Biol. 14:
104-112 (1996) using a method similar to that described in CF and non-C
F Obtain epithelial cells from nasal and bronchial airway epithelial cells. North Carolina University
Excessive donation at the time of lung transplant with the support of the Chapel Hill Association Committee on Human Rights Protection
An incised turbinate or part of the main / lobe bronchi typical of body tissue is obtained.
As described (Wu, R. et al., Am. Rev. Respir. Dis. 13
2: 311-320 (1985)) Target epithelial cells by protease XIV digestion.
Removed from book, but eliminates filtration step. 100mm tissue in modified LHC9 medium
1-2 x 10 per culture dish⁶Place cells. Lechner, J .; F.
And Lavek, M .; A. J. Tiss. Cult. Meth. 9:43
-48 (1985). For modification, EGF concentration was increased to 25 ng / ml,
Acid concentration 5 × 10^-8M, and 0.5 mg / ml bovine serum albumin
And supplementation with 0.8% bovine pituitary extract. About 75% of the overall growth
Cells are harvested by trypsinization and transferred to Transwell Col Insa in modified media.
2.5 × 10 on a plate (Corning Coster, 24 mm diameter, 0.4 μm hole diameter) ^Five One passage was placed at the cell density. LHC Bazaar (Biofluid)
And a 50:50 mixture of DMEM-H were used as bases, with amphotericin and
And gentamicin, except that the EGF concentration was reduced to 0.5 ng / ml.
The land is similar to LHC9 supplemented. Cells were grown to confluency (4 no
After 6 days), air-liquid is applied to the apical surface of the culture for another 25-30 days until use.
Create a physical interface.

Initial histological analysis of human differentiated (WD) cultures derived from non-CF nasal airways after 34 days in culture revealed that epithelial cells were cilia and typical cell types present in human nasal airways in vivo. It is shown to be rich in pseudolamellar mucus gland hair.

Example 2 A 7-cell transmembrane receptor is expressed in the apical membrane of WD cells . A test is performed to identify the 7-TM receptor located in the apical membrane of WD human airway epithelial cells, if any. Cultures of WD cells were exposed to NECA (A _2b receptor agonist), isoproterenol, bradykinin or ATP (all at 10 ^-4 M) and the Cl secretory response was measured. As shown in FIG. 3, a Cl secretory response was detected in the presence of all agonists, but a maximal response was observed in the presence of ATP. These results strongly suggest the presence of functional adenosine, β-adrenergic, bradykinin and purino receptors on the apical surface of airway epithelial cells.

Example 3 AdV Binding and Internalization of Human PD and WD Airway Epithelial Cells AdV Internalization Instead of Adenovirus Vector (AdV) Binding Detects Gene R
The test indicating that the rate limiting step is reducing the efficiency of transfer to the TE WD culture is repeated using the human culture to determine whether the same rate limiting step is present. If this is the case, the efficiency of gene transfer to WD can be increased by increasing the amount of AdV bound to these culture types, even if the rate of cell internalization is reduced. AdV at a defined concentration is poorly differentiated (PD) or differentiated (
WD) Exposure to any of the cultures, approximately 0.1-1% of the total amount of AdV exposed to the cells remains bound after washing. The increase in AdV binding achieved by a single exposure results in an increase in gene transfer, as increased binding of AdV to cells increases the likelihood of internalization leading to AdV entry.

To determine the rate-limiting step of ineffective gene transfer in human cultures, PD and WD cultures were analyzed with ³⁵ S-Ad5VLacZ (A) to analyze AdV binding, internalization and transgene expression in human cultures. 1.2 × ^{10 10} p). A
To determine the effect of increasing the concentration of dV and / or the time of exposure to AdV,
D and WD cultures were incubated at 4 ° C for many time points (1 to 24 hours) in a concentration range (1 to 24 hours).
(10 ⁷ to 10 ¹² p / ml) and exposed to ³⁵ S-Ad5VLacZ (Pick
les, R .; J. Et al., Human Gene Therapy, 7: 921-9.
31 (1996)), after which the cultures were washed in medium and then divided into three groups for analysis. Binding is measured as cell-bound radioactivity. The culture was transferred to 37 ° C. for 6 hours,
The internalization of bound AdV is then determined by removing the radioactivity that has not internalized and then measuring the cell-bound radioactivity. The culture is transferred to 37 ° C. for 48 hours to measure expression and then β-gal. Since the apical surface portion of the cells exposed to the vector is the most appropriate standard and can be directly compared to in vivo epithelial cells, the radioactivity counts per minute (CPM) and β-gal activity can be measured with respect to the nominal surface portion of the culture surface. Standardize.

In PD cells, a 10-fold increase in concentration over a defined incubation time results in a 10-fold increase in AdV binding, internalization, and gene expression, unless receptor uptake and expression system saturation occurs. In WD cultures, a 10-fold increase in AdV binding does not result in a corresponding 10-fold increase in internalization and expression. These data indicate that increased binding alone does not break the rate-limiting step to WD culture (internalization).

[0119] For Example 4 Targeting P2Y ₂ receptor into AdV vectors P2Y ₂ receptor to test the concept of a candidate receptor for targeting based on ability to bind and internalize exogenous ligand, retroviral gene transfer the HA-tagged human P2Y ₂ receptor CHO and A9 cells expressing the (HA tag to the extracellular N-terminal) (not be transduced by AdV both) were obtained. HA-P2Y ₂ receptor expression A9 cells (control cells do not express) stained with fluorescent-labeled anti-HA antibody in static conditions a. Agonist [ATP
γS (10 ^-4 M]] is .P2Y ₂ receptor internalization of approximately 80% of the receptors are internalized within 45 minutes by exposure coated pits (coated pits: co
mediated).

[0120] Next, it was tested whether P2Y ₂ receptors can mediate gene transfer using a bispecific antibody (bs-Ab) research methods. Antibody to influenza hemagglutinin HA. 11 (BabCO) (anti-HA) is directed against the HA epitope to be inserted in the extracellular domain of the human P2Y ₂ receptor expressed in 1321N1 human astrocytoma cells. At neutral pH, the anti-fiber (aneurysm) antibody was treated with m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS).
, Pierce, Rockford, Ill.) Or N- (γ-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBS, Pierce,
(Rockford, Ill.) To make cross-linked antibodies. After reducing and desalting the anti-HA with mercaptoethylamine, the two antibodies are mixed and disulfide bridge formation is possible. The bivalent antibody is purified by continuous chromatography on fiber protein and HA columns.

[0121] Ad fiber (mass) and with this bs-Ab against the HA epitope tag (alpha fibers XarufaHA) of bs-Ab and AdV, binding to HA-P2Y ₂ receptor rather than the null expressing A9 cells was shown . rather than the absence of bs-Ab, the presence of bs-Ab, AdV bound to A9-HA-P2Y ₂ cells.

More importantly, P2Y ₂ receptor-specific gene transfer in A9 and CHO cells was achieved using the bs-Ab approach. In one protocol: (1
B) Different concentrations of bs-Ab (anti-HA / anti-fiber aneurysm; Dr. D. Se from the National Institutes of Health) at 4 ° C. at HA-P2Y ₂ receptor expressing A9 versus null vector expressing A9 cells.
gal's laboratory. R. Pickles), followed by AdV-la
Continuous exposure to cZ (particles per cell = 10 ⁴ ) (Pickles, Rj et al., Human Gene Therapy, 7: 921-931 (1996)); (2) with agonist (ATPγS, 10 ⁻⁴ M) 1 hour incubation at 37 ° C., followed by medium 24 h incubation; and (3) Determination of the gene transfer efficiency by percent measurement and calculation of lacZ-positive cells; using, HA-P2Y ₂ receptor expression A9 cells bs-Ab Is transduced by Ad-lacZ, whereas null A9 cells are not (FIG. 4A). Actually bs-Ab3
The gene transfer efficiency at 0 μg / ml appears to approach nearly 100%.

In a second protocol, the conjugate was used to generate 〜１０10 μg / mlbs-Ab
, An increase in gene transfer reaching a peak was observed (FIG. 4A). The decrease in transduction efficiency at high bs-Ab concentrations may reflect target competition by unbound bs-Ab.

The specificity of gene transfer observed with bs-Abs in A9-HA-P2Y ₂ -R cells was evaluated (FIG. 4B). H exposed to Ad-LacZ and ATPγS
Almost 100% LacZ expression in A-P2Y ₂ -A9 cells was observed. In contrast, either the inappropriate bs-Ab (anti-Ha / anti-ADP1 epitope) or a 40-fold excess of free anti-HA antibody, cells previously exposed to high concentrations of agonist for 24 hours (
Thus, LacZ expression was hardly observed. That HA-P2Y ₂ receptor by these data to mediate gene transfer via the specific interaction with anti-HA bs-Ab evidenced. Finally, similar increase in gene transfer to HA-P2Y ₂ -R A9 cells using bs-Ab and Biotin (αHA / α-biotin) and biotinylated adenovirus vectors for HA was shown.

[0125] Example 5 WD gene transfer efficiency by targeting AdV for the receptor that utilize internalize the mechanism of incorporation of other cells to increase the AdV entry into cultures depends on the internalization efficiency of receptor. To test the internalization efficiency of P2Y ₂ receptor was overexpressed in human P2Y (having extracellular inserted influenza hemagglutinin (HA) epitope tag in the domain) to ₂ receptor 1321N1 human astrocytoma cells. (S. Sromek and T.K.H.
Arden, Molecular Pharmacology, 54, (198
8) Issuance). P2Y ₂ -HA expressing cells that can not be hydrolyzed ATP analog, AT
Incubated with PγS at 37 ° C. After addition of the agonist, the cells were impermeablely fixed with 4% paraformaldehyde at specific time points and washed. Monoclonal anti-HA antibody was incubated with the cells, followed by Cy3-conjugated goat anti-mouse IgG secondary antibody (Jackson Immuno Research Labs). The efficacy of the P2Y ₂ -HA receptor on anti-HA was visualized with a fluorescence microscope. Fix astrocytoma cells in the absence of ATPγS and ATPγS
We were immunostained for the presence of P2Y ₂ -HA receptor after exposure for 30 minutes. When the agonist is present in succession, the immunoreactivity of the plasma membrane and the punctate fluorescent signal are apparently lost, indicating residence in the endosome. Internalization was quantified by an ELISA assay with an efficiency of 80% (receptor site loss 1 hour after ATPγS exposure, Dr. Ken Harden, personal information). This indicates that internalization of the activating receptor is effectively occurring in the astrocytoma cell line.

Example 6 Gene Transfer across the Apical Membrane of Polarized Epithelial Cells Fusion MDCK kidney cells were used as a model for polarized AdV-resistant epithelial cells.
Receptor were localized on the apical surface in MDCK cells expressing HA-P2Y ₂ receptor. Exposing the cells to anti-HA and anti-mouse IgG FITC at 4 ° C.
Receptor localization was measured by confocal microscopy. The HA-P2Y ₂ receptor apical membrane of polarized MDCK cells are desensitized by partially internalization of the receptor was observed. AdV-GFP (continuously compared to neo-expressing MDCK cells)
Treated with green fluorescent protein), Bs directed against HA-P2Y ₂ receptor
-Ab and ATPγS causes transduce HA-P2Y ₂ receptor.
The HA-P2Y ₂ receptor-specific gene transfer as was achieved in AdV resistance cells polarized.

[0127] EXAMPLE 7 Bradykinin II (B2K) receptor expression A9 cells and P2Y ₂ or
Gene Transfer in CHO Cells Expressing or β ₂ Receptors The general applicability of the above approach was demonstrated in other cell types and with other receptors.

A9 cells expressing HA epitope-tagged BK _II receptor and A9 cells expressing neomycin alone were exposed to a bispecific antibody (anti-fiber aneurysm x anti-HA) and AdV in the absence and presence of bradykinin. Briefly, bispecific antibodies (bs-Ab
Incubate the cells at 4 ° C. in the absence or presence of 2 μg at 10 μg / ml), wash, expose to AdVLacZ (10 ¹⁰ particles, 2 hours), wash and wash bradykinin (BK, 1 μM for 2 hours at 37 ° C.). Exposure. The cells were then maintained at 37 ° C. for 24 hours, after which gene expression was assessed by standard techniques. FIG. 5 shows bs-Ab
Only HAB2k expressing cells incubated with both and AdV show effective gene expression with increased gene transfer by receptor activation by agonists.

Little expression was observed with AdV alone, but at very low levels in the presence of AdV + bs-Ab (indicating low levels of receptor turnover even in the absence of ligand). Negligible LacZ expression was observed in null A9 cells regardless of treatment.

In a further study, CHO cells expressing HA epitope-tagged β ₂ adrenoreceptors (Chinese hamster ovary cells lacking the AdV binding receptor)
And wild-type (Wt) CHO cells were continuously exposed to increasing concentrations of bispecific antibody (anti-fiber aneurysm x anti-HA) and AdV and finally isoproterenol.
Briefly, cells are incubated at 4 ° C. in the absence or presence of a bispecific antibody (bs-Ab, 0.1-10 μg / ml for 2 hours), washed and AdvLac
Z (10 ¹⁰ particles, 2 hours), washed, isoproterenol (10 μM,
(2 hours at 37 ° C.). The cells were then maintained at 37 ° C. for 24 hours, after which gene expression was assessed by densitometry of LacZ expressing cells expressed as arbitrary units. Figures 6A B is HA-P2Y ₂ -R (Figure 6A) or HA-
β ₂ (FIG. 6B) shows that gene expression increases in a bs-Ab dose-dependent manner only in CHO cells expressing.

[0131] Example 8 Biotinylated antagonist P2Y ₂ receptor another study method of targeting (or any other 7-TM receptors) relative to the vector be chemically bonded to the modified agonist / antagonist molecules as the target molecule by. The prototype agonist linker is biotin (B) -UTP, which contains a 16 atom linker with the pyrimidine base at position 5 attached to biotin. B-UTP are agonists of P2Y ₂ receptor (Figure 7). P2Y ₂ receptor expression C
If HO cells are exposed to streptavidin-Texas Red (TR) conjugated B-UTP, internalizing the P2Y ₂ receptor B-UTP is a trigger.

In a further experiment, the gene targeted by B-UTP was transformed into HA-P2
It was evaluated stomach Y ₂ receptor expression A9 cells. Were exposed to HA-P2Y ₂ -A9 cells continuously B-UTP, streptavidin (SA) and B-AD (biotinylated adenovirus expression LacZ). Approximately 90% of the HA-P2Y ₂ -A9 cells showed LacZ expression (Fig 8). In contrast, only low levels of LacZ expression were observed in wild-type A9 cells.

Example 9 A biologically active UTP analog has been developed that is more resistant to biological hydrolysis than UTP for binding to dinucleotide agonist vectors. Dinucleotide U ₂ P ₄ is a useful protein. As shown in FIG. 9, U ₂ P ₄ has a function equivalent to UTP in eliciting a biological response (inositol phosphate release) and resists hydrolysis in cystic fibrosis (CF) sputum.

Example 10 Preparation of Bio 16-UP ₄ U

Embedded image The free acid of uridine 5'-monophosphoric acid (1 mmol) (Aldrich, St. Louis, Mo.) is 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 process was repeated one or more times, and the final oil was dissolved in 2.5 ml of dry DMF, to which carbonyldiimidazole (2 mmol) (Aldrich) was added. After stirring at 37 ° C. for 2 hours, the stock solution of activated UMP was reserved. Bio 16-UTP20 vials (250 nmole each) (Boehringer Mannheim) were transferred to one large vial with 100 μl water per vial. The pooled solution was passed through a column (0.5 × 3 cm) of Dowex 50H ⁺ resin (Dow Chemical Company) and flushed with 3 column volumes of water. DMF (
2 ml) and tributylamine (0.25 ml) were added and the solution was evaporated under reduced pressure to give an oil. The oil was dissolved in dry DMF (0.5 ml) and the evaporation was repeated twice. The final oil was dissolved in dry DMF (0.5 ml) and tributylamine (40 μl). Activated UMP solution 200μ prepared above was added to this solution.
1 was added. The reaction mixture was stirred at 37 ° C. for 18 hours and then further activated UMP
The solution (100 μl) was added and the temperature was raised to 50 ° C. After 2 hours at 50 ° C., the reaction mixture was cooled to room temperature and water (0.5 ml) was added. The suspension was filtered through a nylon filter, the solution was concentrated and transferred to an HPLC vial insert. Preparative HPLC (AX300 column; 1M NH ₄ HCO ₃ water / buffer gradient; 4 ml / min) to give the crude reaction mixture, 50% unreacted starting material (Bio 16-UTP) Bio 16-UP ₄ U1 containing .2 mg.

[0135] Example 11 S- biotinylated P ¹ - (4-thiouridine 5 ') - P ⁴ - (uridine 5') tetraphosphate

Embedded image Water (1.0 ml) Medium P ¹ - (uridine ^{5 ') - P 4 - (} 4- thiouridine 5')
A solution of the sodium salt of tetraphosphate (4.825 mol) was stirred with N-iodoacetyl-N'-biotinylhexylenediamine (2.46 mg, 4.825 micromol) at room temperature for 48 hours. The solution was filtered and ion exchange HPLC (Perceptive Biosystems, Poros HQ / H column, 10 aliquots,
(4.6 x 100 mm, gradient 0 to 0.66 M ammonium bicarbonate, 5.0 ml / min) for 20 minutes. Main fraction of each operation (4.7 to 5.4)
Min) were combined and lyophilized P ¹ - (4- (N'- biotinyl amino hexylcarbamoyl methylthiophenyl uridine 5 ') - P ⁴ - (uridine 5') tetraphosphate (Yield 1.426 mmol, 29.6 %, And the absorbance at λ _max 305 nm was compared with the absorbance of a standard solution of 4-methylthiouridine monophosphate to determine the approximate amount.) ³¹ P NMR (D ₂ O, H ₃ PO ₄ , standard) peak Values are δ (ppm) -22.55 (m, 2P) and -10.96 (m, 2P).
The peak value of 1H NMR (D ₂ O, TMS standard) is 1.20 to 1.60 (m, 1
1.4 (s, 2H); 2.08 (t, J = 6.9 Hz, 2H);
59 (d, j = 12.9 Hz, 1H); 2.81 (dd, J = 13.0, 5.0)
Hz, 1H); 2.97 (m, 2H); 3.03 (m, 3H); 3.13 (m, 2H);
1H); 4.04-4.28 (m, 14H); 4.42 (m, 1H); 4.69
(M, partially obscured by _{H 2 O, 10H); 5.78} (m, 4H); 6.6
1 (d, J = 6.6 Hz, 1H); 7.75 (d, J = 8 Hz, 1H); 8.0
8 (d, J = 7.1 Hz, 1H).

[0136] Example ^{11A P 1 - (N 4 -} (6- hexanoate) carbamoyl cytidine 5') P ⁴ - (uridine 5'-) tetraphosphate

Embedded image ^{a) P 1 - (N 4} - ( ethyl-6-hexanoate) carbamoyl cytidine 5 '
-) P ⁴ - (uridine 5'-) tetraphosphate P ¹ - (Cytidine 5 '-) P ⁴ - (uridine 5'-) tetraphosphate, di-butyl ammonium salt (50 mg, 0.432 mmol) in DMF (1 ml )
And tributylamine (10.3 μl, 0.432 mmol) and ethyl-6-isocyanate hexanoate (12.25 μl, 0.648 mmol) was added. The reaction mixture was maintained at 35 ° C. for 4 hours, then at room temperature for 2 days. After the treatment, HPLC purification was performed according to Example 11G to obtain 32 mg (71%), which was calculated as a tetraammonium salt. The peak values of 1H NMR (300 MHz, D ₂ O) are: 8.114 (d, J = 7.2 Hz, 1H), 7.762 (d, J = 7.8H).
z, 1H), 6.429 (d, J = 7.5 Hz, 1H), 5.774 (m, 3H)
), 4.089 (m, 10H), 3.950 (q, J = 7.2 Hz, 2H), 3
. 151 (t, 2H), 2.246 (t, 2H), 1.528 (m, 4H), 1
. 258 (m, 2H), 1.076 (t, J = 7.2 Hz, 3H), 31P (1
21.47 MHz, D2O): -10.09 (m, 2P), -21.73 (m,
2P).

[0137] ^{b) P 1 - (N 4} - (6- hexanoate) carbamoyl cytidine 5 '-) P ⁴
-(Uridine 5'-) tetraphosphate The ethyl ester of the previous step (7 mg, 0.00671 mmol) was dissolved in a mixture of tetrahydrofuran (1 ml) and water (0.5 ml), and lithium hydroxide monohydrate (10 mg, 0 .233 mmol) was added. The mixture was stirred at room temperature for 15 minutes and the base was quenched by adding 1 M hydrochloric acid. The tetrahydrofuran was removed and the product was purified using the same C18 HPLC column and conditions described in Example 11G. 6 mg of the title compound were obtained. (88%) 1H NMR (3
_{00MHz, D 2 O): 8.098} (d, J = 7.5Hz, 1H), 7.742
(D, J = 8.1 Hz, 1H), 6.225 (d, J = 8.1 Hz, 1H), 5
. 777 (m, 3H), 3.132 (t, 2H), 2.198 (t, 2H), 1
. 461 (m, 4H), 1.227 (m, 2H).

[0138] ^{c) P 1 - (N 4} - [6 ((6 '' - biotinamido) hexanamide) hex Nohidorazamido] carbamoyl cytidine 5 '-) P ⁴ - (uridine 5'-) Tet
Rahosufeto previous step of the carboxylic acid (6 mg, 0.00592 mmol) was dissolved in 0.1 M 4-morpholine propane sulfonic acid buffer (1 ml), LC biotin hydrazine (Pierce Chemical, 3 mg, 0.00807 mmol) Added as a solution of dimethyl sulfoxide (150 μl). To this was added 1- [3-dimethylaminopropyl] -3-ethylcarbodiimide hydrochloride (15 μl of a 100 mg / ml solution in 0.1 M 4-morpholinepropanesulfonic acid buffer; 0.0078 mmol) and the solution was stirred at room temperature for 6 hours. did. Further biotin hydrazide (2 mg in 100 μl of dimethyl sulfoxide, 0.00539 mmol) was added and the reaction mixture was stirred for another 2 hours. C18H identical to that described in Example 11G
The product was purified using a PLC column and conditions. 5 mg of the title compound were obtained.
(55%) 1H NMR (300 MHz, D ₂ O): 8.124 (d, J = 7.
2 Hz, 1H), 7.770 (d, J = 7.8 Hz, 1H), 6.255 (d,
J = 7.2 Hz, 1H), 5.799 (m, 3H), 4.450 (m, 1H),
4.190 (m, 10H), 3.161 (t, 2H), 3.046 (t, 2H)
, 2.854 (m, 1H), 2.622 (d, 1H), 2.160 (m, 6H)
, 1.565-1.171 (m, 20H).

[0139] P ¹ - (2 'deoxy -N ⁴ - (ethyl-6-hexanoate) carbamoyl cytidine 5' -) P ⁴ - (uridine 5'-) biotinylated tetraphosphate d) P ¹ - (2'- deoxy -N ⁴ - (ethyl-6-hexa nono benzoate) carbamoyl cytidine 5 '-) P ⁴ - (uridine 5'-) tetraphosphate P ¹ - (2' deoxycytidine 5 '-) P ⁴ - (uridine 5' -) Tetraphosphate, dibutylammonium salt (100 mg, 0.875 mmol) in DMF
(1 ml) and tributylamine (20 μl, 0.841 mmol) and dissolved in ethyl-6-isocyanoate-hexanoate (24.7 μl, 0.131).
Mmol) in one portion. The reaction mixture was heated to 38 ° C. for 3 hours before partitioning between ethyl acetate (2 ml) and water (1 ml). The organic layer was removed and the aqueous layer was washed with ethyl acetate (2x1.5ml). The product was purified by concentrating the aqueous layer to 1 ml and continuously injecting into the same semi-preparative reverse phase column as used in Example 11G. 50 mg (55%) of the title compound were obtained as a tetraammonium salt. 1
1 H NMR (300 MHz, D ₂ O) peak value: 8.074 (d, J = 7)
. 5Hz, 1H), 7.744 (d, J = 8.1 Hz, 1H), 6.221 (d
, J = 7.5 Hz, 1H), 6.089 (t, 1H), 5.748 (m, 2H).
, 4.073 (m, 9H), 3.951 (q, 2H), 3.122 (t, 2H).
, 2.350 and 2.150 (m, 2H), 2.218 (t, 2H), 1.4.
73 (m, 4H), 1.210 (m, 2H), 1.049 (t, 3H), 31P
_{(121.47MHz, D 2 O):} - 10.19 (m, 2P), - 21.83 (
m, 2P).

Following the procedure of steps b to c, the product of step d was reacted with lithium hydroxide / tetrahydrofuran and coupled with LC biotin hydrazine to give the corresponding 2'-deoxycytidine analog.

[0141] Example 11B P ¹ - (2 'deoxy -3, N ⁴ -p- (6- aminohexanoyl amino)-phenyl ethenyl waves * cytidine 5' -) P ⁴ - (uridine 5'-) tetraphosphate bicycloalkyl
Otinization

Embedded image a) 2-Bromo-4 '-(6- bromohexanoyl ) amino-acetophenone 4-Aminoacetophenone (7.36 g, 54.4 mmol) was dissolved in dichloroethane (100 ml) and triethylamine (11.4 ml, 81.4 mmol). 7 mmol) and the solution was cooled to 5-10 ° C. 6-bromohexanoyl chloride (
10 ml, 65.3 mmol) are added in 15 minutes and the heterogeneous suspension is treated with TLC (
Stir for an additional 20 minutes until acylation was indicated by silica gel, 90 chloroform / 10 acetone).

The reaction mixture was treated with 0.5N HCl (75 ml), 50% saturated NaCl (2 × 50
ml), and finally with saturated NaCl (25 ml). Dry the organic layer (
MgSO ₄ ) and evaporated to an oil. The residue was dissolved in toluene (125 ml) while heating to 80-90 ° C., and the solution was left at room temperature for several days.
The crystals obtained were filtered, washed with toluene (5 × 10 ml) and dried at 40 ° C. The yield was 13.08 g (77%). 1H NMR (300 MHz, CD
Cl ₃ ) are: 7.924 (d, 2H), 7.649 (t, 2H),
3.409 (t, 2H), 2.574 (s, 3H), 2.418 (t, 2H),
1.892 (m, 2H), 1.737 (m, 2H), 1.546 (m, 2H).

The product (10.0 g, 32 mmol) was treated with acetic acid (10
0 ml), which was then cooled to 17 ° C. Bromine (2.0 ml, 38.5 mmol) was added via gas tight syringe for 10 minutes and the reaction mixture was stirred at room temperature overnight, at which time TLC (silica gel, 90 chloroform / 10 acetone) left starting material. Not shown. The solution was evaporated to give an orange solid, which was dissolved in chloroform (100 ml). This solution is added with 2% N
Washed with aHSO ₃ (75 ml), 50% saturated NaCL (2 × 50 ml), and finally with saturated NaCL (50 ml). Dry the chloroform layer (MgSO ₄ )
And evaporated and the residue was dissolved in chloroform (100 ml) with heating.
Hexane was added until the solution just became cloudy and it was left at room temperature overnight. The obtained crystals were filtered, washed with 75 chloroform / 25 hexane (2 × 25 ml) and dried at 40 ° C. 5.6 g (45%) of the pure title compound were obtained. 1H NM
The peak value of R (300 MHz, CDCl3) is: 7.955 (d, J = 8.7H)
z, 2H), 7.661 (d, J = 8.7 Hz, 2H), 7.654 (s, 1H)
), 4.418 (s, 2H), 3.414 (t, 2H), 2.426 (t, 2H).
), 1.922 (m, 2H), 1.851 (m, 2H), 1.541 (m, 2H)
).

[0144] b) P ¹ - (2'- deoxy -3, N ⁴ -p- (6- bromohexanoyl amino) phenylethenyl * waves * cytidine 5 '-) P ⁴ - (uridine 5'-) tetraphosphate
DOO P ¹ - (2 'deoxycytidine 5' -) P ⁴ - (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) in DMF (2 ml
) Solution was added and the resulting solution was heated at 42 ° C overnight. The pH was adjusted to 7 by addition of 1M sodium bicarbonate and the reaction mixture was partitioned between ethyl acetate (10ml) and water (15ml). The aqueous layer was extracted once more with ethyl acetate (10 ml),
Evaporation gave a foam. The crude was dissolved in water (1 ml) and 400 μl was applied to a semi-preparative reversed-phase column (Altec Absorbent Sphere Nucleotide / Nucleotide C18, 10 × 250 mm, 7 μm; 0.1 M ammonium acetate to methanol gradient for 30 minutes) 5 ml / min; monitor observation at 295 nm)
Purified by continuous injection into. Appropriate fractions were pooled, evaporated and lyophilized to give 18 mg (34%) of the title compound as a tetraammonium salt. 1H NMR (300 MHz, D ₂ O): 7.72 (s, 1H), 7.616
(M, 2H), 7.456 (d, 8.4 Hz, 2H), 7.253 (d, 7.8
Hz, 2H), 6.646 (d, 7.8 Hz, 2H), 6.266 (t, 1H)
, 5.615 (m, 2H), 4.084 (m, 9H), 3.351 (t, 2H).
, 2.267 (m, 4H), 1.727 (m, 2H), 1.536 (m, 2H)
1.334 (m, 2H).

[0145] c) P ¹ - (2 'deoxy -3, N ⁴ -p- (6- aminohexanoyl amino) phenylethenyl * waves * cytidine 5' -) P ⁴ - (uridine 5 ') from tetraphosphate previous step Bromide (16 mg, 0.0141 mmol) was dissolved in concentrated ammonium hydroxide (2 ml) and stirred at room temperature overnight. The reaction mixture was evaporated to give a foam, reconstituted in water (500 μl) and the same semi-preparative C as used in the previous step
The product was purified by several consecutive injections under the same conditions on 18 columns. Evaporate, lyophilize and 10 mg (66 mg) of the title compound as the tetraammonium salt.
%). The peak value of 1H NMR (300 MHz, D ₂ O) is: 7.636
(D, J = 8.4 Hz, 1H), 7.563 (s, 1H), 7.472 (d, J
= 8.4 Hz, 1H), 7.319 (d, J = 8.1 Hz, 2H), 7.142
(D, J = 8.7 Hz, 2H), 6.491 (d, J = 7.8 Hz, 1H), 6
. 299 (t, 1H), 5.646 (t, 2H), 4.085 (m, 9H), 2
. 859 (t, 2H), 2.298 (m, 3H), 2.150 (m, 1H), 1
. 554 (m, 4H), 1.306 (m, 2H).

D) P ^1- (2′-deoxy-3, N ⁴ -p- [6-((6 ″ -biotinamide) hexanamido) hexanoyl] aminophenylethenocytidine 5 ′-) P ⁴
-(Uridine 5'-) tetraphosphate The amine from the previous step (7 mg, 0.000654 mmol) was dissolved in water (1 ml) and succinimidyl-6- (biotinamide) hexanoate (Pierce Chemicals, EZ-Link) was added. NHS-LC-biotin, 15 mg, 0.0
329 mmol) in DMF (2 ml) was added. The pH was adjusted to 8.5 by the addition of 1M sodium bicarbonate and the mixture was stirred at room temperature for 2 hours. The solvent was removed and the product was purified by several consecutive injections on HPLC using the same semi-preparative C18 column and conditions as in the previous step. 7 mg (76%) of the title compound were obtained as the tetraammonium salt. The peak values of 1H NMR (300 MHz, D ₂ O) are: 7.876 (s, 1H), 7.615 (m, 4H), 7.351 (d, J =
7.8 Hz, 2H), 6.714 (d, J = 7.8 Hz, 2H), 6.355 (
t, 1H), 5.651 (m, 2H), 4.321 (t, 1H), 4.094 (
m, 9H), 3.029 (t, 2H), 2.930 (m, 1H), 2.817 (
t, 2H), 2.715 (m, 1H), 2.517 (d, 1H), 2.288 (
m, 4H), 1,978 (t, 2H), 1.882 (t, 2H), 1.544 (
m, 2H), 1.5-0.9 (m, 23H). 31P NMR (121.47 MHz, D2O): -10.56 (m, 2P)
, -21.859 (m, 2P).

[0147] Example 11C P ¹ - (6- thioinosine 5 '-) P ⁴ - (uridine 5') Tetoraho S- biotinylated Sufeto

Embedded image P in water (0.5ml)¹-(6-thioinosine 5 ')-P^Four-(Uridine 5 ')
A solution of the sodium salt of tetraphosphate (8 mg, 8.7 μmol) was added to N
-Iodoacetyl-N'-biotinylhexylenediamine (5.5 mg, 11
(Micromol) at room temperature for 4 hours. Filter the solution and in 13 aliquots
Ion exchange HPLC (Perceptive Biosystems, Poros HQ / H color
4.6 x 100 mm, 0 to 0.66 M ammonium bicarbonate for 20 minutes
Gradient (5.0 ml / min). Major fractions from each operation (2.
48 to 2.93 minutes), freeze-dried and¹-(6- (N'-bioti)
Nylaminohexylcarbamoylmethylthioinosine 5 ')-P^Four-(Uridine
5 ') Tetraphosphate was obtained (yield 7.07 mg, 5.52 micromol,
63.5%).³¹P NMR (D_TwoO, H_ThreePO_FourThe peak value of the standard is: δ (pp
m) -21.82 (m, 2P), 10.24 (m, 2P). 1H NMR (D _Two O, TMS standard): 0.87 (m, 4H), 1.06 (m, 4H), 1.17
(M, 2H), 1.375 (m, 4H); 1.98 (t, J = 7.0 Hz, 2H
); 2.47 (d, J = 13.1 Hz, 1H), 2.67 (dd, J = 13.0)
, 4.9 Hz, 1H), 2.82 (m, 2H); 2.95 (m, 3H); 3.8
6 (s, 2H); 4.03 (m, 8H); 4.17 (m, 1H); 4.33 (m
, 2H); 4.55 (m, partially H_TwoO. Unclear, 1H); 4.65 (m
, Partially H_Two5.58 (d, J = 8.2 Hz, 1H)
); 5.63 (d, J = 4.5 Hz, 1H); 5.97 (d, J = 5.7 Hz,
7.5H (d, J = 8.0 Hz, 1H); 8.49 (s, 1H);
53 (s, 1H).

Example 12 Study of AdV Internalization Process in Human PD and WD Cultures It has been reported that α _v β _3/5 integrin mediates internalization but does not mediate the binding of Ad to epithelial cells in vitro. I have. W of membrane-bound α _v β _3/5 integrin
Localization of D cultures to the apical and / or basolateral membranes is important in understanding the role of these molecules in Adv-mediated gene transfer. The efficacy of many different antibodies against these integrins places integrins on polarized epithelial cells to be measured. These integrins are also receptors for RGD amino acid sequence containing peptides. Numerous studies have shown that the RGD peptide inhibits AdV-mediated gene transfer to epithelial cells by interacting with α _v β _3/5 integrin. This example describes the effect of RGD peptide on AdV binding internalization and transgene expression in PD and WD cultures.

Rat bronchial epithelial cell (RTE) differentiation initially localizing α _v β _3/5 integrin (
WD) Integrin antibody-specific immunoprecipitation has been developed using cultures. Antibodies to these integrins are commercially available; therefore, antibodies (R838, gift from Dr. Steven Albelda of the University of Pennsylvania, PA)
Was used and raised against the human epithelial cell vitronectin receptor, which has been shown to cross-react with rat α _v β _3/5 integrin ³⁷ . Easy WD
Either the apical or basolateral domain of the culture was exposed to sulfo-NHS-biotin (0.5 mg / ml, Pierce) at 4 ° C. to biotinylate only the outer membrane proteins. After solubilizing the cells in a non-denaturing lysis buffer (in the presence of a protease inhibitor), the proteins were immunoprecipitated and separated by Western analysis on a 4-12% acrylamide gel (Novex) under non-reducing conditions. Biotinylated proteins were probed with a secondary antibody to peroxidase conjugated to streptavidin and detected by ECL analysis (Super Signal CL-HRP, Pierce). Biotinylated proteins identified by R838 in shown FIG. 5 (corresponding to the band of 125 kD alpha _v, 97 kD is beta _3/5 subunit), although present in the basolateral membrane and HeLa cells RTE cells, WD Apparently not present in the apical membrane of the cultures, suggesting that the rat vitronectin receptor is not located at the apical end of these culture types. The absence of these integrins in the apical membrane is due to the low rate of AdV internalization into these cultures.

Example 13 Identification and localization of integrins present in human PD and WD cultures Apical and / or basolateral membrane proteins of human PD and WD cultures were coated at 4 ° C. on individual surfaces with sulfo-NHS-biotin. Selectively isolated by exposure. Selective human antibody _{(LM609, α v β 3;} P1F6, α v β 5; VNR147,
A standard immunoprecipitation experiment is performed with α _v ; P4G11, β ₁ ; CD61, β ₃ ; anti-β ₅ and R838, α _v β _3/5 ; all obtained from Chemicon, Inc., California). This method allows for the localization and detection of α _v β integrin in the apical and / or basolateral membrane.

Example 14 Interference of AdV Internalization by RGD Peptides As described above, adenovirus binding, internalization and transgene expression in PD and WD cultures in the absence and presence of RGD peptides are measured. Hexapeptide, biologically active GRGDSP (Gibco-BRL) and inactive control peptide GRGESP (Gibco-BRL) at final concentrations of 0.1 to 4.
0 mg / ml at 4 ° C. for 2 hours, added to PD and WD cultures, followed by AdV (1
0 ¹⁰ p / ml). Cyclic RGD peptide (Immunodynamics, La Jolla, CA) has been reported to more strongly reduce AdV-mediated gene transfer and is also used. Analysis of AdV binding, internalization and transgene expression is performed as described above.

Example 15 Study of the cellular uptake process for AdV entry The initial binding of AdV to epithelial cells is via a fiber (nodule) protein. It is not clear whether the fiber protein alone is sufficient to induce internalization and endosomal formation, or whether the role of the fiber is to assist in the placement of the virus and the use of intrinsic endocytosis. However, the α _v β _3/5 integrin mediates somewhat the internalization of AdV into the cytoplasm. T. J. Wickham et al., Cell, 73: 309-319 (1
993). Α _v in the apical membrane of both WD cultures and cartilage airway epithelial cells
It has been speculated that the absence or low number of β _3/5 integrins (MJG).
Oldman et al. Virol. 69: 5951-5958 (1995)),
It is likely that both internalization rates and gene transfer efficiencies are reduced in these cell types. Therefore, the cellular uptake process that may be responsible for the entry of AdV into cells is studied. First, in order to understand the functional role of fiber (nodule) protein-cell interaction, the nodule protein was bound to fluorescent microspheres of the same diameter as Ad, and the nodules in PD and WD cells were examined by confocal microscopy.
Assess the first interaction of the spheres. Increased specific or non-specific binding can increase internalization and possibly expression. Second, human Arufabuibeta ₅ integrins overexpressed in WD cultures, directing the protein to the apical membrane.
This is used to test the hypothesis that α _v β ₅ expression in the apical membrane is internalized and therefore the rate-limiting step in gene expression. Third, to understand the cell entry pathways utilized by Ad, we test cell lines that are defective or functional in coated pit receptor-mediated endocytosis. We test the assumption that high concentrations of AdV can utilize non-specific entry pathways to access cells. Finally, use of other cellular uptake mechanisms to increase the efficiency of AdV internalization into WD cells, ie, to target AdV to specific receptor types that undergo endocytosis when stimulated by an exogenous ligand. Test your strategy.

Example 16 Adenovirus entry into AdV internalizing cells into the HeLa cell line in functional and defective receptor-mediated endocytosis can be achieved by a number of cellular pathways: receptor-mediated endocytosis by coated pits, non-specific pinoviruses. Cytosis or phagocytosis (RM Steinman et al., J. Cell Biol. 96: 1-2).
7) influences the uptake. The role of coated pit-mediated endocytosis and non-specific pinocytosis in AdV invasion into functional or defective receptor-mediated endocytic cell lines is studied. Wild-type dynamin protein or its mutant form, mDyn (regulated by TET-inducible promoter)
HeLa cell mutants capable of overexpressing either of the above were produced. (H. Damke et al., J. Cell Biol., 127: 915-934) Normally, dynamin contributes to coated pit endosomal formation and function by "pinching" depressions in the plasma membrane. HeLa cells overexpressing wild-type dynamin show no functional or morphological changes in the uptake process compared to parental cells. Cells overexpressing mDyn form coated pits and sink the cytoplasmic membrane, but coated vesicles do not extend into the cytoplasm. Receptor-mediated endocytosis (EGF and transferrin)
The ligand for is that cells expressing mDyn cannot internalize,
Ligand-receptor binding, coated pit aggregates, receptor recruitment to coated pits, and depression of the plasma membrane are all unaffected. In the absence of receptor-mediated endocytosis, non-specific pinocytosis initially remains unchanged, but is eventually upregulated to compensate for the loss of receptor-mediated endocytosis.

Example 17 Incorporation process for entry of AdV into HeLa cells HeLa cells overexpressing either wild-type or mDyn (Scripps Research Institute, La Jolla, CA, Sando)
ra Schmid) is used to study the uptake process predominant in AdV entry into these cells. The AdV concentration range is studied to determine whether high titers of AdV lead to cellular uptake by non-specific processes. Briefly, a monolayer of wild-type or mutant HeLa cells expressing mdyn dynamin was grown on plastic and Ad5VLacZ (10 ⁶ -10 ¹¹ iu / ml, MOI at 4 ° C. for 2 hours).
~ 1 to 10 ⁵ ) and then 0 to 24 hours at 37 ° C. without washing
At which point the cells are washed and maintained at 37 ° C. for 48 hours before performing the β-gal enzyme assay. The differences in gene expression observed in the two cell lines at defined time points reflect the participation of receptor-mediated endocytosis in AdV entry. In addition, comparative studies will also be performed using fluorescent microspheres (with and without binding knob proteins) to reveal the interaction of these proteins with specific and non-specific uptake processes.

Example 18 Analysis of Transgene Expression in Human PD and WD Cultures Virus titers in a defined range on either the apical and / or basolateral membrane (10 ⁶ to 10 ¹¹ infectious units / ml; MOI range 1 to 10) ^{At 5} ), the PD and WD cultures are exposed to Ad5VLacZ with increasing exposure times (1 to 24 hours), and the resulting concentration and time on gene expression are tested. The vector used in this test is produced and titered by the UNC gene therapy core. Incubations are performed at 37 ° C and / or 4 ° C. The former temperature allows the function of the cellular uptake process to be tested, while the latter temperature is a standard technique for measuring the initial binding of a ligand to its receptor in the absence of receptor recycling and / or internalization. is there. Gene expression is assessed 48 hours after initial exposure to AdV by both qualitative and quantitative means (X gal histochemical and standard colorimetric enzyme assays, respectively). Pickles, R.A. J. Et al., Human Gene
See Therapy, 7: 921-931 (1996).

Although the invention has been described with respect to specific embodiments thereof, it will be understood that the invention allows for further modifications. This application is intended to cover any adaptations, uses, or adaptations of the invention generally in accordance with the principles of the invention, which are applicable to the essential features set forth in the scope of the above-described aspects of the invention. It is intended to include such departures from the present disclosure as are known or customary in the relevant arts.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a virus-receptor complex of the present invention. This figure shows adenovirus targeted to an internalizing 7-cell transmembrane receptor.

FIG. 2 is an illustration of a particular embodiment of the present invention.

FIG. 3 is an illustration of the Cl ⁻ secretory response of human airway epithelium to luminal NECA (A _2b agonist), isoproterenol, bradykinin, or ATP (all 10 ⁻⁴ M).

FIG. 4A shows sequential (open circles) or preformed conjugates (closed triangles)
FIG. 5 is a graphic illustration of the dose-effect relationship between bs-Ab concentration and gene transfer efficiency in A9-null cells (closed circles) compared to HA-P2Y ₂ -A9 administered as. Figure 4B is a specific or pre-treated with nonspecific bs-Ab, or after chronic desensitization of HA-P2Y ₂ receptor by pretreatment with ATPγS, A9-HA-P
Figure ₂ is an illustration of a study to evaluate the specificity of increased gene transfer with bs-Ab in 2Y2 and A9-null cells.

FIG. 5 is an illustration of gene transfer using bs-Ab into A9 cells expressing null A9 and HA-tagged BK _II receptor.

FIG. 6 is an illustration of gene transfer in CHO cells using a bs-Ab for HA-tagged P2Y ₂ and β ₂ receptors and adenovirus fiber proteins.

FIG. 7 shows stimulation of inositol phosphate formation by biotin-UTP in astrocytoma cells expressing the P2Y ₂ receptor (closed circles) but not wild type (closed squares). It is an illustration.

FIG. 8 is an illustration of stimulation of gene transfer in A9 (wt) and HA-P2Y ₂ -A9 cells in the presence of biotin-UTP conjugated to biotin-Ad with streptavidin.

FIG. 9A is an illustration comparing the agonist strength of U ₂ P ₄ and UTP in astrocytomas expressing the P2Y ₂ receptor. FIG. 9B is an illustration of the metabolic stability of U ₂ P ₄ compared to UTP in cystic fibrosis sputum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/08 C07K 16/28 4H045 16/28 C12N 15/00 A (81) Designated countries EP (AT, BE , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C U, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM Pickles, Raymond Jay 27576, North Carolina, USA, Chapel Hill, Smith Avenue 420 (72) Inventor Ride, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW Out, Janet El, Morningside Drive, Raleigh, North Carolina, USA 27607 3101 (72) Inventor Pendergast, William 27713, North Carolina, USA Durham, Williamsburg Way 5815 (72) Inventor Yaksa, Benjamin Earl 27608, North Carolina, USA 1330 Raleigh, Hathaway Road 1330 (72) Inventor Douglas, James G., The Third 27502, North Carolina, Apex, USA Charrion Downs Rain 2204 F-term (reference) 4B024 AA01 BA63 CA04 DA02 EA02 FA11 FA20 GA11 GA18 HA13 HA17 4C057 BB02 CC01 MM05 MM09 4C084 AA13 ZA59 ZC80 4C085 AA21 4C087 AA10 BC83 4H045 EA11

Claims (52)

[Claims] 1. A method for delivering a heterologous nucleic acid to a cell, comprising contacting a conjugate with said cell, said conjugate comprising a transfer vector and a ligand, wherein said transfer vector is delivered to said cell. Wherein said ligand specifically binds to a G protein-coupled receptor and said cell is under conditions that allow said vector to be internalized into said cell.
The G protein-coupled receptor is expressed, and the ligand comprises a nucleotide represented by any of the following formulas I to III, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable ester or salt thereof: The method selected from the group consisting of: Formula I is: Wherein X is oxygen, methylene, difluoromethylene, imide, n is any of 0, 1, or 2, m is any of 0, 1, or 2, and n + m = 0, 1, 2, 3, or 4; and B and B ′ are each independently either a purine residue or a pyrimidine residue linked through position 9 or 1. Z = OH or N ₃ , Z ′ = OH or N ₃ , Y = H or OH, Y ′ = H or OH, provided that when Z is N ₃ , Y is H Or Y ′ is H when Z ′ is N ₃ , and Formula II is Wherein R ₁ , X ₁ , X ₂ and X ₃ are each, independently, O ⁻ or S ⁻ , R ₅ and R ₆ are H, while R ₇ is none. , N-3 and C-4 have a double bond (cytosine), or R ₅ , R ₆ and R ₇ together represent —CH ＝ CH—, and N-4 and C—
4 has a double bond, forms a ring from N-3 to N-4, and is optionally substituted at the 4- or 5-position of the etheno ring (3, N ⁴ -ethenocytosine) , Formula III: Wherein R ₁ , X ₁ , X ₂ and X ₃ are defined as in Formula I, and R ₃ and R ₄ are H
While R ₂ is empty and there is a double bond between N-1 and C-6 (adenine), or R ₃ and R ₄ are H, while R ₂ is O. And there is a double bond between N-1 and C-6 (adenine 1-oxide), or R ₃ , R ₄ and R ₂ together are —CH ＝ CH— and N-6 And C-
6 has a double bond, forms a ring from N-6 to N-1, and is optionally substituted at the 4- or 5-position of the etheno ring (1, N6-ethenoadenine). 2. The method according to claim 1, wherein the compound of formula I is a compound of formula Ia. Formula Ia is: Wherein X = O, n + m = 1 or 2, Z, Z ′, Y, and Y ′ = OH, and B and B ′ are defined by formulas Ib and Ic; Ib is: Wherein R ₂ is O or nothing, or R ₁ and R ₂ together can form an optionally substituted 5-membered fused imidazole ring; or R ₃ R ₁ or 8-NHR ₃ group of ₁ group HNR includes (a) a arylalkyl aryl moiety is optionally substituted (C ₁ ~ ₆₎ group, and (b) alkyl, (c) and ([6-aminohexyl] carbamoylmethyl), and (d) .omega.-aminoalkyl (C ₂ ~ _10), and (e) .omega.-hydroxyalkyl _{(C 2 ~ 10), (} f) ω- thiol alkyl ( a C ₂ ~ _10), and (g) .omega.-carboxyalkyl (C ₂ ~ _10), and (h) (b), any of the .omega.-acylated derivatives of (c), or (d), where Is an acetyl, trifluoroacetyl, benzoyl, or substituted benzoyl group. Is either Ruarukiru of (C ₂ ~ _10), and (i) .omega.-carboxyalkyl such as those described above _{(e) (C 2 ~ 10} ), the carboxyl moiety herein is an ester or amide, consisting Selected from the group: Formula Ic is , And the formula, R ₄ is hydroxy, mercapto, amino, cyano, aralkoxy, a C ₁ ~ ₆ alkylthio, C ₁ ~ ₆ alkoxy, C ₁ ~ ₆ alkylamino or dialkylamino, wherein the dialkylamino here alkyl groups may form a heterocyclic ring linked optionally, R ₅ is hydrogen, acyl, C ₁ ~ ₆ alkyl, aroyl, is any one of C ₁ ~ ₆ alkanoyl, benzoyl or sulfonate, R ₆ is hydroxy, mercapto, alkoxy, aralkoxy, is any one of C ₁ ~ ₆ alkylthio, C ₁ ~ ₅ disubstituted amino, triazolyl, alkylamino or dialkylamino, alkyl groups of said dialkylamino here, or form a heterocyclic ring optionally linked or substituted optionally linked to N ³ , Ureido or form or a substituted ureido, ureido N atoms away from the pyrimidine ring here is substituted with C ₁ ~ ₆ alkyl group or C ₆ ~ ₁₀ aryl group or a C ₇ ~ ₁₂ arylalkyl group, It is, where alkyl, aryl and arylalkyl moieties, amino, hydroxy, thio, carbonyl or sulfonyl substituents (also themselves be substituted) may be substituted with any, or, R ₅ to R ₆ are combined Te, to form a 5-membered or 6-membered saturated or unsaturated ring bonded through N or O of R _6, wherein said ring is optionally substituted, R ₇ is (a) a hydrogen (B) hydroxy, (c) cyano, (d) nitro, and (e) alkenyl, wherein the alkenyl moiety is optionally linked through oxygen and is adjacent to oxygen. Forming a ring optionally substituted with an alkyl or aryl group on carbon, (f) substituted alkynyl, (g) halogen, (h) alkyl, (i) substituted alkyl, (j) and perhalomethyl, and (k) C ₂ ~ ₆ alkyl, (l) and C ₂ ~ ₃ alkenyl, (m) and substituted ethenyl, (n) and C ₂ ~ ₃ alkynyl, (o) R ₆ is amino or substituted amino In other cases, R ₈ is selected from the group consisting of: substituted alkynyl; and R ₈ is (a) hydrogen, (b) alkoxy, (c) arylalkoxy, (d) alkylthio, and (e) arylalkylthio. (F) carboxamidoethyl, (g) carboxymethyl, (h) methoxy, (i) methylthio, (j) phenoxy, and (k) phenylthio. Wherein the substituted derivative of adenine is any of adenine 1-oxide, 1, N6- (4 or 5-substituted etheno) adenine, 6-substituted adenine, or 8-substituted aminoadenine, wherein 6- or 'radicals R' 8-HNR includes arylalkyl aryl moiety is optionally functionalized (C ₁ ~ ₆₎ group, and the alkyl, ([6-aminohexyl] carbamoylmethyl) -, and ω -Acylated-amino (hydroxy, thiol and carboxy) derivatives wherein the acyl group is
Acetyl, trifluoroacetyl, benzoyl or substituted benzoyl, wherein the carboxyl moiety is present as an ethyl or methyl ester derivative thereof, or a methyl, ethyl or benzamide derivative thereof). And an alkyl group having 3. The method according to claim 1, wherein the compound of formula I is a compound of formula Id. Formula Id is: It is. Wherein X is any of oxygen, methylene, difluoromethylene, or imide, n = 0 or 1, m = 0 or 1, and n + m = 0, 1
Or B and B ′ are each independently linked via position 9 or 1; a purine residue as in formula Ib as described in claim 2; A pyrimidine residue as in formula Ic, wherein B and B
'Is uracil attached to the ribosyl moiety at the N-1 position, then the sum of m + n is 3 or 4 when X is oxygen. 4. The method of claim 1, wherein the furanose sugar of Formula I is in either the β-D configuration, or the D configuration, or the L configuration, or the D and L configurations. 5. The method according to claim 1, wherein said vector is a viral vector. 6. The vector may be an adenovirus vector, an adeno-associated virus vector, a human retrovirus vector, a non-human retrovirus vector,
The method according to claim 1, which is a viral vector selected from the group consisting of a herpes virus vector. 7. The method of claim 7, wherein said viral vector is selected from the group consisting of a lentivirus vector and a Moloney murine leukemia virus vector. 8. The method according to claim 1, wherein said vector is an oligonucleotide. 9. The method according to claim 1, wherein said ligand is an antibody. 10. The method according to claim 1, wherein said ligand is a peptide. 11. The method of claim 1, wherein said ligand is selected from the group consisting of nucleotides, nucleosides, catecholamines, C5A, and bradykinin. 12. The method of claim 1, wherein said ligand is selected from the group consisting of a G protein coupled receptor agonist and a G protein coupled receptor antagonist. 13. The method of claim 1, wherein said conjugate is a covalent conjugate. 14. The method of claim 1, wherein said cells are airway epithelial cells. 15. The method of claim 1, wherein said cells are differentiated columnar airway epithelial cells. 16. The method of claim 1, wherein said contacting is performed in vitro. 17. The method of claim 1, wherein said contacting is performed in vivo. 18. The method of claim 1, wherein said conjugate is formed before said contacting step. 19. A bispecific antibody having a first binding region that specifically binds to a viral vector and a second binding region that specifically binds to an extracellular epitope of a G protein-coupled receptor. 20. A conjugate useful for delivering a heterologous nucleic acid to a cell, wherein the conjugate comprises a transfer vector and a ligand, wherein the transfer vector comprises a heterologous nucleic acid to be delivered to the cell, wherein the ligand comprises , Which specifically bind to a G protein-coupled receptor, wherein the ligand is
A conjugate selected from the group consisting of the nucleotides represented by Formula I through Formula III of 21. The conjugate according to claim 20, wherein said vector is a viral vector. 22. The vector according to claim 20, wherein the vector is a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a human retrovirus vector, a non-human retrovirus vector, and a herpes virus vector. Conjugate. 23. The conjugate of claim 20, wherein said vector is a viral vector selected from the group consisting of a lentivirus vector and a Moloney murine leukemia virus vector. 24. The conjugate of claim 20, wherein said ligand is an antibody. 25. The conjugate according to claim 20, wherein said ligand is a peptide. 26. The conjugate of claim 20, wherein said ligand is selected from the group consisting of nucleotides, nucleosides, catecholamines, C5A, and bradykinin. 27. The conjugate of claim 20, wherein said ligand is selected from the group consisting of a G protein coupled receptor agonist and a G protein coupled receptor antagonist. 28. The conjugate of claim 20, wherein said conjugate is a covalent conjugate. 29. A method comprising contacting a conjugate with a cell, the conjugate comprising a transfer vector and a ligand, wherein the transfer vector comprises a heterologous nucleic acid to be delivered to the cell, and wherein the ligand comprises a G protein A method for delivering a heterologous nucleic acid, which specifically binds to a coupled receptor and expresses the G protein-coupled receptor, to the cell under conditions that allow the cell to internalize the vector into the cell. 30. The method according to claim 29, wherein said vector is a viral vector. 31. The vector according to claim 29, wherein the vector is a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a human retrovirus vector, a non-human retrovirus vector, and a herpes virus vector. the method of. 32. The viral vector is selected from the group consisting of a lentivirus vector and a Moloney murine leukemia virus vector.
The vector according to claim 1. 33. The method according to claim 29, wherein said vector is an oligonucleotide. 34. The method of claim 29, wherein said ligand is an antibody. 35. The method of claim 29, wherein said ligand is a peptide. 36. The method of claim 29, wherein said ligand is selected from the group consisting of nucleotides, nucleosides, catecholamines, C5A, and bradykinin. 37. The method of claim 29, wherein said ligand is selected from the group consisting of a G protein coupled receptor agonist and a G protein coupled receptor antagonist. 38. The method of claim 29, wherein said conjugate is a covalent conjugate. 39. The method of claim 29, wherein said cells are airway epithelial cells. 40. The cell of claim 29, wherein the cell is a differentiated cylindrical airway epithelial cell.
The method described in. 41. The method of claim 29, wherein said contacting is performed in vitro. 42. The method of claim 29, wherein said contacting is performed in vivo. 43. The method of claim 29, wherein said conjugate is formed prior to said contacting step. 44. The conjugate comprises a transfer vector and a ligand, wherein the transfer vector comprises a heterologous nucleic acid to be delivered to the cell, wherein the ligand binds the heterologous nucleic acid specifically binding to a G protein-coupled receptor to the cell. Conjugates useful for delivery. 45. The conjugate of claim 44, wherein said vector is a viral vector. 46. The vector according to claim 44, wherein the vector is a viral vector selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a human retrovirus vector, a non-human retrovirus vector, and a herpes virus vector. Conjugate. 47. The conjugate of claim 44, wherein said vector is a viral vector selected from the group consisting of a lentivirus vector and a Moloney murine leukemia virus vector. 48. The conjugate of claim 44, wherein said ligand is an antibody. 49. The conjugate of claim 44, wherein said ligand is a peptide. 50. The conjugate of claim 44, wherein said ligand is selected from the group consisting of nucleotides, nucleosides, catecholamines, C5A, and bradykinin. 51. The conjugate of claim 44, wherein said ligand is selected from the group consisting of a G protein coupled receptor agonist and a G protein coupled receptor antagonist. 52. The conjugate of claim 44, wherein said conjugate is a covalent conjugate.