DETAILED DESCRIPTION OF THE INVENTION

[0019] In one aspect, the present invention provides compositions and methods to enhance the delivery of nucleic acids to tissues. In one embodiment, the method provides contacting the cells of the tissue or organ with a gene delivery system comprising an interferon gene in conjunction with delivery enhancing agent.

[0020] Gene Delivery System:

[0021] A “gene delivery system” comprises a recombinant polynucleotide encoding an interferon gene operatively linked to expression control sequences to effect expression of the interferon gene in a cell.

[0022] The term polynucleotide refers to a polymer composed of nucleotide monomers. Polynucleotides include naturally the occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleic acid analogs include those non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidites, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “nucleic acid” typically refers to large polynucleotides. The term “oligonucleotide” typically refers to short polynucleotides, generally less than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.” “Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell of the subject. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, e.g., a “recombinant interferon polypeptide.”

[0023] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an interferon amino acid sequence or polypeptide” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0024] “Expression control sequence” refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and/or translation) of a nucleotide sequence operatively linked thereto. “Operatively linked” refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence). Expression control sequences can include, for example and without limitation, sequences of promoters (e.g., inducible or constitutive), enhancers, transcription terminators, a start codon (i.e., ATG), splicing signals for introns, and stop codons.

[0025] An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

[0026] In one embodiment, a particular expression control sequence may employed to provide selective expression of the interferon gene in a particular type of tissue by the use of a promoter and/or other expression elements preferentially used by the tissue of interest. Examples of known tissue-specific promoters include the promoter for creatine kinase, which has been used to direct the expression of dystrophin cDNA expression in muscle and cardiac tissue (Cox et al., Nature, 364:725-729 (1993)); immunoglobulin heavy or light chain promoters for the expression of genes in B cells; albumin or alpha-fetoprotein promoters to target cells of liver lineage and hepatoma cells, respectively. Exemplary tissue-specific expression elements for the liver include, but are not limited, to HMG-CoA reductase promoter (Luskey, Mol. Cell. Biol., 7(5):1881-1893 (1987)); sterol regulatory element 1 (SRE-1; Smith et al., J. Biol. Chem., 265(4):2306-2310 (1990); phosphoenol pyruvate carboxy kinase (PEPCK) promoter (Eisenberger et al., Mol. Cell Biol., 12(3):1396-1403 (1992)); human C-reactive protein (CRP) promoter (Li et al., J. Biol. Chem., 265(7):4136-4142 (1990)); human glucokinase promoter (Tanizawa et al., Mol. Endocrinology, 6(7):1070-81 (1992); cholesterol 7-alpha hydroylase (CYP-7) promoter (Lee et al., J. Biol. Chem., 269(20):14681-9 (1994)); beta-galactosidase alpha-2,6 sialyltransferase promoter (Svensson et al., J. Biol. Chem., 265(34):20863-8 (1990); insulin-like growth factor binding protein (IGFBP-1) promoter (Babajko et al., Biochem Biophys. Res. Comm., 196 (1):480-6 (1993)); aldolase B promoter (Bingle et al., Biochem J, 294(Pt2):473-9 (1993)); human transferrin promoter (Mendelzon et al., Nucl. Acids Res., 18(19):5717-21 (1990); collagen type I promoter (Houglum et al., J. Clin. Invest., 94(2):808-14 (1994)). Exemplary tissue-specific expression elements for the prostate include, but are not limited to, the prostatic acid phosphatase (PAP) promoter (Banas et al., Biochim. Biophys. Acta., 1217(2):188-94 (1994); prostatic secretory protein of 94 (PSP 94) promoter (Nolet et al., Biochim. Biophys. ACTA, 1098(2):247-9 (1991)); prostate specific antigen complex promoter (Casper et al., J. Steroid Biochem. Mol. Biol., 47 (1-6):127-35 (1993)); human glandular kallikrein gene promoter (hgt-1) (Lilja et al., World J. Urology, 11(4):188-91 (1993). Exemplary tissue-specific expression elements for gastric tissue include, but are not limited to, the human H + /K + -ATPase alpha subunit promoter (Tanura et al., FEBS Letters, 298:(2-3):137-41 (1992)). Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, pancreatitis associated protein promoter (PAP) (Dusetti et al., J. Biol. Chem., 268(19):14470-5 (1993)); elastase 1 transcriptional enhancer (Kruse et al., Genes and Development, 7(5):774-86 (1993)); pancreas specific amylase and elastase enhancer promoter (Wu et al., Mol. Cell. Biol., 11(9):4423-30 (1991); Keller et al., Genes & Dev., 4(8):1316-21 (1990)); pancreatic cholesterol esterase gene promoter (Fontaine et al., Biochemistry, 30(28):7008-14 (1991)). Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter (Helftenbein et al., Annal. NY Acad. Sci., 622:69-79 (1991)). Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, cholesterol side-chain cleavage (SCC) promoter (Rice et al., J. Biol. Chem., 265:11713-20 (1990). Exemplary tissue-specific expression elements for the general nervous system include, but are not limited to, gamma-gamma enolase (neuron-specific enolase, NSE) promoter (Forss-Petter et al., Neuron, 5(2):187-97 (1990)). Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF—H) promoter (Schwartz et al., J. Biol. Chem., 269(18):13444-50 (1994)). Exemplary tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-1/granzyme B promoter (Hanson et al., J. Biol. Chem., 266 (36):24433-8 (1991)); the terminal deoxy transferase (TdT), lambda 5, VpreB, and Ick (lymphocyte specific tyrosine protein kinase p56lck) promoter (Lo et al., Mol. Cell. Biol., 11(10):5229-43 (1991)); the humans CD2 promoter and its 3′ transcriptional enhancer (Lake et al., EMBO J., 9(10):3129-36 (1990)), and the human NK and T cell specific activation (NKG5) promoter (Houchins et al., Immunogenetics, 37(2):102-7 (1993)). Exemplary tissue-specific expression elements for the colon include, but are not limited to, pp60c-src tyrosine kinase promoter (Talamonti et al., J. Clin. Invest, 91(1):53-60 (1993)); organ-specific neoantigens (OSNs), mw 40 kDa (p40) promoter (Ilantzis et al., Microbiol. Immunol., 37(2):119-28 (1993)); colon specific antigen-P promoter (Sharkey et al., Cancer, 73(3 supp.) 864-77 (1994)). Exemplary tissue-specific expression elements for breast cells include, but are not limited to, the human alpha-lactalbumin promoter (Thean et al., British J. Cancer., 61(5):773-5 (1990)).

[0027] The use of the term “interferon gene” refers to a gene that directs the expression of an interferon.

[0028] The term “gene” as used herein is intended to refer to a nucleic acid sequence which encodes an polypeptide. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not affect the function of the gene product. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. Nucleic acid sequences encoding the polypeptide can be DNA or RNA which directs the expression of a specific protein or peptide. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full length sequences derived from the full-length protein. It is further understood that the sequence includes the degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific host cell.

[0029] The term interferon as used herein is intended to include all classes and subclasses of interferon polypeptides, and deletion, insertion, or substitution variants, including conservative amino acid substitutions, thereof, biologically active polypeptide fragments thereof, and allelic forms thereof. The term “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides. “Conservative substitution” refers to the substitution in a polypeptide of an amino acid with a functionally or structurally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:

[0030] 1) Alanine (A), Serine (S), Threonine (T);

[0031] 2) Aspartic acid (D), Glutamic acid (E);

[0032] 3) Asparagine (N), Glutamine (O);

[0033] 4) Arginine (R), Lysine (K);

[0034] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0035] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0036] The term “biologically active” as used herein refers to any anti-viral or anti-proliferative or anti cancer activity as measured by techniques well known in the art (see, for example, Openakker et al., supra; Mossman, J. Immunol. Methods, 65:55 (1983)).

[0037] “Allelic form” refers to any of two or more polymorphic forms of a polypeptide occupying the same genetic locus. Allelic variations arise naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. “Allelic forms” also refer to cDNAs polypeptides derived from mRNA transcripts of genetic allelic variants.

[0038] As used herein, the term “alkyl” denotes branched, unbranched, or cyclic hydrocarbon substituent or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent substituents, having the number of carbon atoms designated (i.e. C 1 -C 10 means one to ten carbons) Examples of saturated hydrocarbon substituents include, but are not limited to, groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, octa-decyl, 2-methylpentyl, cyclohexyl, (cyclohexyl)methyl, cyclopentylmethyl. The substituents can be optionally substituted with one or more functional groups which are attached commonly to such chains, such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto, or thio, cyano, alkylthio, aryl, heteroaryl, carboxyl, nitro, amino, alkoxyl, amido, and the like to form alkyl substituents such as carboxymethyl, trifluoromethyl, 3-hydroxyhexyl, 2-carboxypropyl, and the like. An unsaturated alkyl substituent is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The substituents can be substituted with one or more functional groups which are attached commonly to such chains as described for saturated hydrocarbons.

[0039] The term “aryl” means a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.

[0040] The term “heteroaryl” refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. The above noted aryl and heteroaryl ring systems can be further substituted with one or more functional groups which are attached commonly to such ring systems such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, carboxyl, nitro, amino, alkoxyl, or amido.

[0041] The term “acyl” denotes the —C(O)R— substituent, wherein R is alkyl or aryl as defined above, such as but not limited to benzoyl, succinyl, acetyl, propionyl or butyryl.

[0042] The term “hydroxyl” denotes the substituent —OH—.

[0043] The term “alkoxy” denotes the substituent —OR— where R is alkyl.

[0044] The term “amino” denotes an amine linkage (—NRR′) where R and R′ are independently hydrogen substituent, alkyl substituent, or aryl substituent.

[0045] The term “carboxylate” denotes the substituent —OC(O)R—, wherein R is an optionally substituted alkyl or aryl.

[0046] The term “acyloxy” denotes the substituent —(CRR′) m C(O)OR″—, wherein R and R′ are independently selected from a group comprising of an alkyl substituent, aryl substituent or hydrogen substituent and R″ is hydrogen or an alkyl substituent and m is an integer between 1-8, inclusive.

[0047] The term “halogen” or “halo” refers to the substituents F, Cl, Br, or I.

[0048] The term “saccharide residue” refers to a monosaccharide substituent which can include more than one monosaccharide substituent linked as a homo-oligosaccharide substituent (an oligosaccharide comprising one type of monosaccharide) or hetero-oligosaccharide substituent (an oligosaccharide comprising more than one type of monosaccharide). In a preferred embodiment, the homo and hetero-oligosaccharide substituent is composed of 2 to 10 monosaccharide units. Monosaccharides can include pentose or hexose residues and the residues can exist as the cyclized or uncyclized (open-chain) form. When a monosaccharide is in the open chain form, the oxygen atom of the carbonyl carbon can be replaced with —RR′— where R and R′ are independently selected from a group of an alkyl substituent, a halogen substituent, a hydroxyl substituent, a hydrogen substituent, a amino substituent, or a alkoxy substituent. Preferred oligo-saccharides include a pentose-pentose disaccharide group, a hexose-hexose disaccharide group, a pentose-hexose disaccharide group, and a hexose pentose disaccharide group. The monosaccharide can be selected from a group of ribose, arabinose, xylose and lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, or talose, whereby one or more the hydroxyl groups on the monosaccharide can be replaced with hydrogen, alkyl substituent, alkoxy substituent, amino substituent, or an acyl substituent.

[0049] A variety of interferon polypeptides are known to those of ordinary skill in the art. In some embodiments, the interferon polypeptide is Type I or Type II interferon, including those commonly designated as alpha-interferon, beta-interferon, gamma-interferon, and omega-interferon (e.g., α-interferon, β-interferon, γ-interferon and {acute over (ω)}-interferon), and combinations thereof, including the consensus sequence for alpha-interferon. In some embodiments, the alpha-interferon is alpha 1 or alpha 2 -interferon. In some embodiments, the gene encodes interferon α-2b.

[0050] In some embodiments, the interferon gene is that of a wild-type interferon polypeptide. In other embodiments, the interferon gene encodes an interferon polypeptide that is substantially identical to a wild-type interferon polypeptide or a fusion protein thereof. The terms “identical” or percent “identity,” in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence. In the context of two nucleic acids or polypeptides, the term “substantially identical” refers to two or more sequences or subsequences that have at least 70% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0051] In other embodiments, the interferon polypeptide encoded by the gene delivery system is substantially identical to the sequence of a native polypeptide over a stretch of 50 amino acids, 100 amino acids, 150 amino acids or the entire length of the native polypeptide. In some embodiments, the interferon polypeptide encoded by the gene delivery system is 99%, 98%, 95% or 90% identical to the sequence of a wild-type interferon polypeptide over a stretch of 50 amino acids, 100 amino acids, or the entire length of the native polypeptide. In some embodiments, the interferon polypeptide encoded by the gene delivery system is substantially identical to the sequence of a human wild-type alpha-interferon polypeptide over a stretch of 50 amino acids, 100 amino acids, or the entire length of the native polypeptide. In some embodiments, the interferon polypeptide encoded by the gene delivery system is 99%, 98%, 95% or 90% identical to the sequence of the human wild-type polypeptide over a stretch of 50 amino acids, 100 amino acids, 150 amino acids or the entire length of the native polypeptide.

[0052] In some embodiments, the interferon is a hybrid interferon. The construction of hybrid alpha-interferon genes containing combinations of different interferon subtype sequences (e.g., α and Δ, α and β, and α and F) is disclosed in U.S. Pat. Nos. 4,414,150, 4,456,748, and 4,678,751. U.S. Pat. Nos. 4,695,623, 4,897,471 and 5,831,062 disclose novel human leukocyte interferon polypeptides having amino acid sequences which include common or predominant amino acids found at each position among naturally-occurring alpha interferon subtype polypeptides and are referred to as consensus human leukocyte interferon. In one embodiment of the invention, the hybrid interferon is interferon α2 α1.

[0053] In one embodiment, the interferon is an interferon-α. Recombinant interferon alphas, for instance, have been cloned and expressed in E. coli (e.g., Weissmann et al., Science, 209:1343-1349 (1980); Sreuli et al., Science, 209:1343-1347 (1980); Goeddel et al., Nature, 290:20-26 (1981); Henco et al., J. Mol. Biol., 185:227-260 (1985)). In some embodiments, the interferon is a human interferon alpha. In some embodiments, the interferon alpha is interferon alpha 2a or 2b (see, for example, WO 91/18927).

[0054] The gene delivery system generally is provided in the form of a eucaryotic expression vector capable of directing the expression of the interferon gene in a eucaryotic cell. Examples of eucaryotic expression vectors include viral and non-viral vectors. The term “eucaryotic expression vector” refers to viral and non-viral vectors prepared by conventional recombinant DNA techniques comprising interferon expression cassette. The term “expression cassette” is used herein to define a nucleotide sequence capable of directing the transcription and translation of a interferon coding sequence comprising expression control elements operably linked to a interferon coding sequence so as to result in the transcription and translation of a the interferon sequence in a transduced mammalian cell. A variety of viral and non-viral delivery vectors useful to achieve expression of nucleotide sequences in transduced cells are known in the art. See, e.g. Boulikas, T in Gene Therapy and Molecular Biology, Volume I (Boulikas, T. Ed.) 1998 Gene Therapy Press, Palo Alto, Calif. pages 1-172

[0055] Examples of non-viral delivery systems used to introduce the interferon gene to a target cell include expression plasmids capable of directing the expression of the interferon. Expression plasmids are autonomously replicating, extrachromosomal circular DNA molecules, distinct from the normal genome and nonessential for cell survival under nonselective conditions capable of effecting the expression of a DNA sequence in the target cell. The expression plasmid may also contain promoter, enhancer or other sequences aiding expression of the therapeutic gene and/or secretion can also be included in the expression vector. Additional genes, such as those encoding drug resistance, can be included to allow selection or screening for the presence of the recombinant vector. Such additional genes can include, for example, genes encoding neomycin resistance, multi-drug resistance, thymidine kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase.

[0056] The expression plasmid containing the interferon gene may be encapsulated in liposomes. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. The delivery of nucleic acids to cells using liposome carriers is well known in the art. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. Ann. Rev. Biophys. Bioeng. 9:467 (1980), Szoka, et al. U.S. Pat. No. 4,394,448 issued Jul. 19, 1983, as well as U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. Liposomes useful in the practice of the present invention may be formed from one or more standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. Examples ofsuch vesicle forming lipids include DC-chol, DOGS, DOTMA, DOPE, DOSPA, DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP, n-spermidine cholesterol carbamate and other cationic lipids as disclosed in U.S. Pat. No. 5,650,096. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. Additional components may be added to the liposome formulation to increase serum half-life such as polyethylene glycol coating (so called “PEG-ylation”) as described in U.S. Pat. No. 5,013,556 issued May 7, 1991 and U.S. Pat. No. 5,213,804 issued May 25, 1993.

[0057] In order to provide directed delivery of the non-viral interferon gene delivery system to a particular cell, it may be advantageous to incorporate elements into the non-viral delivery system which facilitate cellular targeting. For example, a lipid encapsulated expression plasmid may incorporate modified surface cell receptor ligands to facilitate targeting. Although a simple liposome formulation may be administered, the liposomes either filled or decorated with a desired composition of the invention of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Examples of such ligands includes antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, chimeric antibodies or functional fragments (Fv, Fab, Fab′) thereof. Alternatively, the DNA constructs of the invention can be linked through a polylysine moiety to a targeting moiety as described in Wu, et al. U.S. Pat. No. 5,166,320 issued Nov. 24, 1992 and Wu, et al, U.S. Pat. No. 5,635,383 issued Jun. 3, 1997.

[0058] In one embodiment of the invention as exemplified herein, the vector is a viral vector. The terms virus(es) and viral vector(s) are used interchangeably herein. The viruses useful in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxyiridae, adenoviridiae, or picornnaviridiae. The viral genomes may be modified by conventional recombinant DNA techniques to provide expression of interferon and may be engineered to be replication deficient, conditionally replicating or replication competent. Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al.(1997) Nature Biotechnology 15:866-870) may also be useful in the practice of the present invention. Minimal vector systems in which the viral backbone contains only the sequences needed for packaging of the viral vector and may optionally include an interferon expression cassette may also be employed in the practice of the present invention. In some instances it may be advantageous to use vectors derived from different species from that to be treated which possess favorable pathogenic features such as avoidance of pre-existing immune response. For example, equine herpes virus vectors for human gene therapy are described in WO98/27216 published Aug. 5, 1998. The vectors are described as useful for the treatment of humans as the equine virus is not pathogenic to humans. Similarly, ovine adenoviral vectors may be used in human gene therapy as they are claimed to avoid the antibodies against the human adenoviral vectors. Such vectors are described in WO 97/06826 published Apr. 10, 1997.

[0059] Many viruses exhibit the ability to infect a broad range of cell types. However, in some applications it may be desirable to infect only a certain subpopulation of cells. Consequently, a variety of techniques have evolved to facilitate selective or “targeted” vectors to result in preferential infectivity of the mature viral particle of a particular cell type. Cell type specificity or cell type targeting may also be achieved in vectors derived from viruses having characteristically broad infectivities such as adenovirus by the modification of the viral envelope proteins. For example, cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fiber coding sequences to achieve expression of modified knob and fiber domains having specific interaction with unique cell surface receptors. Examples of such modifications are described in Wickham, et al. (1997) J. Virol. 71(11):8221-8229 (incorporation of RGD peptides into adenoviral fiber proteins); Arnberg, et al. (1997) Virology 227:239-244 (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Harris and Lemoine (1996) TIG 12(10):400-405; Stevenson, et al. (1997) J. Virol. 71(6):4782-4790; Michael, et al.(1995) gene therapy 2:660-668 (incorporation of gastrin releasing peptide fragment into adenovirus fiber protein); and Ohno, et al. (1997) Nature Biotechnology 15:763-767 (incorporation of Protein A-IgG binding domain into Sindbis virus). Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins (see, e.g. Michael, et al. (1993) J. Biol. Chem. 268:6866-6869, Watkins, et al. (1997) Gene Therapy 4:1004-1012; Douglas, et al. (1996) Nature Biotechnology 14: 1574-1578. Viral vectors encompassing one or more of such targeting modifications may optionally be employed in the practice of the present invention to enhance the selective infection and expression of interferon in lung tissues, especially epithelial tissues of the lung.

[0060] In one embodiment of the invention as exemplified herein, the vector is an adenoviral vector. The term adenoviral vector refers collectively to animal adenoviruses of the genus mastadenovirus including but no limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F sugenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A and Ad 11P), 12, 13,14,15,16,17,18,19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. The term bovine adenoviruses includes, but is not limited to, bovine adenovirus types 1,2,3,4,7, and 10. The term canine adenoviruses includes but is not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2. The term equine adenoviruses includes, but is not limited to, equine types 1 and 2. The term porcine adenoviruses includes but is not limited to porcine types 3 and 4. The use of adenoviral vectors for the delivery of exogenous transgenes are well known in the art. See e.g., Zhang, W-W. (1999) Cancer Gene Therapy 6:113-138. In one embodiment the adenoviral vector for expression of the interferon sequence is a replication deficient human adenovirus of serotype 2 or 5 created by elimination of adenoviral E1 genes resulting in a virus which is substantially incapable of replicating in the target tissues, optionally including a deletion of the protein IX function. A preferred recombinant viral vector is the adenoviral vector delivery system which has a deletion of the protein IX gene. See such systems disclosed in U.S. Pat. No. 6,210,939 which is assigned to the same assignee as the present invention and is herein incorporated by reference in its entirety for all purposes.

[0061] Preferred vectors are derived from the adenoviral, adeno-associated viral and retroviral genomes. In the most preferred practice of the invention, the vectors are derived from the human adenovirus genome. Preferred vectors are derived from the human adenovirus serotypes 2 or 5. The replicative capacity of such vectors may be attenuated (to the point of being considered “replication deficient”) by modifications or deletions in the E1a and/or E1b coding regions. Other modifications to the viral genome to achieve particular expression characteristics or permit repeat administration or lower immune response are preferred.

[0062] Alternatively, the viral vectors may be conditionally replicating or replication competent. Conditionally replicating viral vectors are used to achieve selective expression in particular cell types while avoiding untoward broad spectrum infection. The viral genome may be modified to include inducible promoters which achieve replication or expression only under certain conditions. Examples of inducible promoters are known in the art (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; lida, et al. (1996) J. Virol. 70(9):6054-6059; Hwang, et al.(1997) J. Virol 71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al.(1997) J. Biol. Chem. 272(46); 29364-29371. The viruses may also be designed to be selectively replicating viruses such as those described in Ramachandra, et al. PCT International Publication No. WO 00/22137, International Application No. PCT/US99/21452 published Apr. 20, 2000 and Howe, J., PCT International Publication No. WO WO0022136, International Application No. PCT/US99/21451 published Apr. 20, 2000. The virus may also be modified to be attenuated for replication in certain cell types. For example the adenovirus dl1520 containing a specific deletion in the E1b55K gene (Barker and Berk (1987) Virology 156: 107) has been used with therapeutic effect in human beings. Such vectors are also described in McCormick (U.S. Pat. No. 5,677,178 issued Oct. 14, 1997) and McCormick, U.S. Pat. No. 5,846,945 issued Dec. 8, 1998.

[0063] The vectors of the present invention may be modified to encode an additional transgene to the interferon gene, either in tandem through the use of IRES elements or through independently regulated promoters.

[0064] The present invention provides a delivery enhancing compound that, when formulated with the interferon gene delivery system enhances the delivery of the interferon gene to the target cell, tissue, or organ. “A delivery-enhancing compound” or “delivery enhancing agent” refers to any compound that enhances delivery of the interferon gene or gene delivery system to a cell, tissue or organ. Enhanced delivery as used herein refers to either or both of an increase in the number of copies of the interferon gene or gene delivery system that enter each cell or a increase in the proportion of cells in, for example, a tissue or organ, that take up the interferon gene or interferon gene delivery system. Administration of an interferon gene delivery system to a tissue or organ in conjunction with a delivery enhancing compound results in an increase in the amount of the interferon gene that is delivered and expressed within the cells, relative to the amount of the gene delivered and expressed in the cells when administered in the absence of the delivery enhancing compound. The determination of whether a particular compound is effective in enhancing delivery of a nuclei acid delivery system by means known to those of skill in the art. To assess translation and protein expression, a reporter gene such as beta-galactosidase or green fluorescent protein may be incorporated into the nucleic acid delivery system to produce a readily assayable signal to assess the level of enhanced gene expression. To assess transcription and replication, the presence of copies of DNA may be assessed by PCR analysis.

[0065] Certain delivery enhancing compounds may possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention. Single diastereomer of pairs of enantiomers, for example, can be obtained by fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active acid as a resolving agent. Alternatively, any enantiomer of an inventive compound may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.

[0066] Delivery enhancing compounds may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed by the inventive formulas.

[0067] The delivery enhancing compounds may have unnatural ratios of atomic isotopes at one or more of their atoms. For example, the compounds may be radiolabeled with isotopes, such as tritium or carbon-14. All isotopic variations of the compounds of the present invention, whether radioactive or not, are within the scope of the present invention. Where it is described in a compound formula that a “group” or “moiety” is attached to another portion of the compound, it is to be understood that a radical corresponding to the “group” or “moiety” wherein a H atom has been removed to form the radical is meant. The delivery enhancing compounds may be isolated in the form of their pharmaceutically acceptable acid addition salts, such as the salts derived from using inorganic and organic acids. Such acids may include hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, malonic and the like. In addition, certain compounds containing an acidic function can be in the form of their inorganic salt in which the counterion can be selected from sodium, potassium, lithium, calcium, magnesium and the like, as well as from organic bases. The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.

[0068] Exemplary delivery-enhancing compounds are taught in U.S. Pat. No. 6,165,779, and U.S. patent application Ser. No. 08/889,355, filed on Jul. 8, 1997 and U.S. patent application Ser. No. 09/650,359, filed on Aug. 28, 2000, which are each assigned to the same assignee as the present application and are incorporated by reference in their entireties. Such compounds include, but are not limited to, detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates. Alcohols include, for example, the aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols include, for example, glycerin, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol. Acetates such as acetic acid, gluconic acid, and sodium acetate are further examples of delivery enhancing compounds. Hypertonic salt solutions such as 1M NaCl are also examples of delivery enhancing compounds. Examples of surfactants include sodium dodecyl sulfate (SDS) and lysolecithin, polysorbate 80, nonylphenoxy-polyoxyethylene, lysophosphatidylcholine, polyethyleneglycol 400, polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO. Bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other astringents like silver nitrate can also be used, as can heparin-antagonists like quaternary amines such as protamine sulfate. Cyclooxygenase inhibitors such as, for example, sodium salicylate, salicylic acid, and non-steroidal anti-inflammatory drugs (NSAIDS) such as indomethacin, naproxen, and diclofenac are also suitable.

[0069] Detergents that can function as delivery enhancing compounds include, for example, anionic, cationic, zwitterionic, and nonionic detergents. Exemplary detergents include, but are not limited to, taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride, ZWITTERGENT®3-14 detergent, CHAPS (3-[(3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate, hydrate, Aldrich), Big CHAP, Deoxy Big CHAP, TRITON®-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC®-F68 detergent, TWEEN® 20 detergent, and TWEEN® 80 detergent (CALBIOCHEM® Biochemicals).

[0070] One example of a preferred delivery enhancing compound is, for example, a nucleic acid is Big CHAP, which is a cholate derivative (see, e.g., Helenius et al. (I 979) “Properties of Detergents,” In: Methods in Enzymology, Vol. 66, 734-749. Especially preferred delivery enhancing compounds are compounds of Formulae, I, II, III, IV, V, or VI and their pharmaceutically acceptable salts.

[0071] In additional embodiments, the invention provides compositions comprising the interferon gene delivery system and at least one delivery enhancing compound that has a Formula I: 1

[0072] in which X 1 is and X 2 are selected from the group consisting of 2

[0073] and X 3 is a saccharide group wherein the saccharide group can be selected from the group consisting of pentose monosaccharide groups, hexose monosaccharide groups, pentose-pentose disaccharide groups, hexose-hexose disaccharide groups, pentose-hexose disaccharide groups, and hexose-pentose disaccharide groups. Other saccharide groups can include more than one monosaccharide linked in either homo-oligosaccharides or hetero-oligosaccharides. Preferred monosaccharides include pentose and/or hexose residues. For example, the saccharide groups can be selected from the group consisting of pentose monosaccharide groups, hexose monosaccharide groups, pentose-pentose disaccharide groups, hexose-hexose disaccharide groups, pentose-hexose disaccharide groups, and hexose-pentose disaccharide groups. One example of a preferred saccharide group for X 3 is lactose.

[0074] In some embodiments, the delivery enhancing compounds of Formula I have X 3 saccharide groups that are composed of three or more monosaccharides. Preferably, the saccharide group has between one and eight monosaccharides, more preferably between one and four monosaccharides, and most preferably about two to three monosaccharides. The use of a trisaccharide, for example, can provide a compound having increased solubility.

[0075] In a further embodiment, X 1 and X 2 are both 3

[0076] and X 3 is a glucose group.

[0077] Exemplary delivery enhancing compounds for use according to the invention include compounds of Formula II: 4

[0078] in which R 1 and R 2 are each independently a member selected from the group consisting of hydrogen, and a hydroxyl group; m and n are each independently selected from about 0-2; R 3 is selected from the group consisting of —NR 4 R 5 wherein R 4 and R 5 are each independently a member selected from the group consisting of a hydrogen, a saccharide residue, an optionally substituted alkyl, an optionally substituted acyl, and an optionally substituted acyloxy, and a quaternary ammonium salt —NR 6 R 7 R 8 X wherein R 6 , R 7 and R 8 are independently a member selected from the group consisting of hydrogen and C 1 -C 4 alkyl, and X is the negatively charged ionically bound counterion selected from the group consisting of pharmacologically acceptable counterions. In some embodiments, the counter ion is a halogen or an optionally substituted carboxylate.

[0079] In one embodiment, the exemplary compound of Formula II has R 1 and R 2 which are both hydroxyl groups. In another embodiment, the compound of Formula II is one in which m and n each equal to 1. In another embodiment, the compound of Formula II is one in which R 4 is hydrogen; and R 5 is a member selected from the group consisting of a hydrogen, a saccharide residue, an optionally substituted alkyl, an optionally substituted acyl, and an optionally substituted acyloxy.

[0080] In one embodiment, the delivery enhancing compound has formula VI: 5

[0081] In another embodiment, the delivery enhancing compound has Formula VII (SYN3): 6

[0082] In another embodiment, at least one of R 4 or R 5 is succinyl or acetyl. In another embodiment, R 3 is a trimethylammonium salt.

[0083] In another embodiment, R 4 and R 5 are each independently a member selected from the group consisting of a hydrogen, an optionally substituted alkyl, an optionally substituted acyl, and an optionally substituted acyloxy, and a quaternary ammonium salt —NR 6 R 7 R 8 X wherein R 6 , R 7 and R 8 are independently a member selected from the group consisting of hydrogen and C 1 -C 4 alkyl, and X is the negatively charged ionically bound counterion selected from the group consisting of pharmacologically acceptable counterions.

[0084] One example of a preferred delivery enhancing compound for use in the methods and compositions is SYN3, which has Formula VII. Methods of making SYN3 and its homologues are taught in U.S. Pat. No. 6,392,069 which is assigned to the same assignee as the present application and incorporated by reference in its entirety.

[0085] For some applications, it is desirable to use delivery enhancing compounds in the methods and compositions of the invention that exhibit increased water solubility and/or delivery enhancing activity compared to other compounds. Exemplary delivery enhancing compounds are of Formula I in which R is a cationic group. Suitable cationic groups include, for example, tetramethyl and ammonium moieties, and salts thereof.

[0086] In another aspect, the invention provides a pharmaceutical formulation for administration of a recombinant adenovirus encoding an interferon gene and a delivery enhancing agent. In some embodiments, the delivery enhancing agent comprises SYN3 or a SYN3 homolog. In some embodiments, the gene delivery system comprises about 10 9 -10 12 particles (PN)/ml recombinant adenovirus encoding an interferon gene, about 0.1 to 10 mg/ml SYN3, a SYN3 solubilizing agent (e.g., a surfactant, including Big CHAP or hydroxy propyl beta cyclodextrin (HPβCG) or TRITON™-X-100 detergent/ an octylphenoxypolyethoxy ethanol), a buffer and pharmacologically acceptable excipients. In some embodiments, the interferon gene is an alpha-interferon, beta-interferon, gamma-interferon, or omega-interferon gene or a fusion thereof or a portion thereof encoding a polypeptide with an interferon anti-cancer, anti-infective, or immune system modulating bioactivity. In one embodiment, the amount of SYN3 in the formulation is 1-10 mg/ml. The compositions may be lyophilized for dilution with a suitable pharmaceutical carrier prior to use.

[0087] In some embodiments, the invention also provides a formulation that contains the interferon gene delivery system and a delivery enhancing compound. The concentration of the delivery enhancing compound in a formulation will depend on a number of factors such as the particular gene deliver system and delivery enhancing compound being used, the buffer, pH, target tissue or organ and mode of administration. The concentration of the delivery enhancing compound will depend substantially upon the agent used. For agents effective at higher concentrations, the concentrations will often be in the range of 1% to 50% (v/v), preferably 10% to 40% (v/v) and most preferably 15% to 30% (v/v). For delivery enhancing agents effective at lower concentrations, the particular delivery enhancing compound of the invention may be preferably used in the range of about 0.002 to 2 mg/ml, more preferably about 0.02 to 2 mg/ml, most preferably about 0.1 to 1 mg/ml in the formulations of the invention. The delivery enhancing compounds of the invention which comprise SYN3 or its homologs or analogs are preferably used in the range of about 0.002 to 20 mg/ml, more preferably about 0.02 to 2 mg/ml, most preferably about 0.1 to 1 mg/ml in the formulations of the invention.

[0088] The delivery enhancing compounds for use in the methods and compositions of the invention are typically formulated in a solvent in which the compounds are soluble, although formulations in which the compounds are only partially solubilized are also suitable. Phosphate buffered saline (PBS) is one example of a suitable solubilizing agent for these compounds, and others are known to those of skill in the art. One will recognize that certain additional excipients and additives may be desirable to achieve solubility characteristics of these agents for various pharmaceutical formulations. For example, well known solubilizing agents such as detergents, fatty acid esters, and surfactants can be added in appropriate concentrations so as to facilitate the solubilization of the compounds in the various solvents to be employed. Where the formulation includes a detergent, the detergent concentration in the final formulation administered to a patient is preferably about 0.5-2×the critical micellization concentration (CMC). Suitable detergents include those listed above. The identification of suitable detergents and appropriate concentrations for their use can be determined as described herein. One example of a solubilizing agent for compounds such as SYN3 and related compounds is Tween 80 at a concentration of approximately 0.05% to about 0.3%, more preferably at a concentration of about 0.10% to about 0.15%. Big CHAP is also a solubilizing agent for SYN3 and related compounds.

[0089] When used for pharmaceutical purposes, the formulations of the invention include a buffer that contains the delivery-enhancing compound. The buffer can be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to the ordinarily skilled artisan such as those described by Good et al., Biochemistry, (1966) 5:467. The pH of the buffer in the pharmaceutical composition comprising a modulatory gene contained in an adenoviral vector delivery system, for example, is typically in the range of 6.4 to 8.4, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0090] The compositions of this invention can additionally include a stabilizer, enhancer or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the recombinant adenoviral vector delivery system comprising the tumor suppressor gene. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would know that the choice of pharmaceutically acceptable carrier depends on the route of administration and the particular physio-chemical characteristics of the recombinant adenoviral vector delivery system and the particular tumor suppressor gene contained therein. Examples of carriers, stabilizers or adjuvants can be found in Martin, Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton, Pa. 1975), and Remington: The Science and Practice of Pharmacy ( 19th Ed) (Lippincott, Williams & Wilkins Publisher, December 1995) which are incorporated herein by reference.

[0091] An exemplary formulation for administration of a recombinant adenovirus is about 10 9 -10 11 PN/ml virus, about 0.1 to 1 mg/ml SYN3 with a suitable SYN3 solubilizing agent (e.g., about 2-10 mM Big CHAP or about 0.1-1.0 mM TRITON®-X-100) detergent, in phosphate buffered saline (PBS), plus about 2-3% sucrose (w/v) and about 1-3 mM MgCl ₂ , at about pH 6.4-8.4.

[0092] The present invention also provides such pharmaceutical compositions comprising SYN3 in combination with a pharmaceutically acceptable carrier. The carrier may be aqueous or nonaqueous. The composition may include at least one pharmaceutically acceptable solubilizer. The composition may be lyophilized pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier, at least one pharmaceutically acceptable solubilizer and a at least one pharmaceutically acceptable bulking agent.

[0093] A further aspect of the invention is a pharmaceutical composition comprising an interferon gene delivery system and SYN3 in combination with a pharmaceutically acceptable aqueous carrier, at least one pharmaceutically acceptable solubilizer, and at least one pharmaceutically acceptable bulking agent.

[0094] Such pharmaceutical compositions may further comprise SYN3 in combination with a pharmaceutically acceptable carrier and an expression vector or gene delivery system comprising an interferon DNA sequence inserted into the vector to which the interferon DNA is foreign. The nucleic acid sequence of the interferon may, in some embodiments, may be foreign to or not otherwise significantly expressed in the intended target tissue or organ of the subject. See U.S. patent application Ser. No. 10/329,043 [titled “SYN3 COMPOSITIONS AND METHODS” Attorney Docket No. 016930-000841US] filed on Dec. 20, 2002 and assigned to the same assignee as the present application and hereby incorporated by reference in its entirety.

[0095] Solvents that may be used for the formulations of the present invention include, for example, aqueous solvents such as water for injection, and/or nonaqueous solvents, such as DMSO and N,N-Dimethyylacetamide, also known as DMA, and co-solvent mixtures, e.g., glycerol and water, as prepared preferably in accordance with USP standards.

[0096] The formulations preferably contain polysorbates, or polyoxyethylene sorbitan esters, a class of nonionic surfactants that included, e.g., polysorbate 80 and polysorbate 20, amongst others, Pluronics, or polyethylenepolypropylene glycol block copolymers, a class of nonionic surfactants, that include, e.g., Pluronic L68 and L92, amongst others, and hydroxypropyl-beta-cyclodextrin, a polysubstituted hydroxyalkyl-beta-cyclodextrin, which is a class of nonionic complexing agents, that include, e.g., HPβCD and Big CHAP. Preferred are HPβCD, Big CHAP, Polysorbate 80, Polysorbate 20, Pluronic L64, and Pluronic L92 as solubilizing agents. The solubilizers can be used, for example, either individually or in combinations. The concentrations of the solubilizing agents are set forth below. HPβCD can be present in a concentration of about 50 to 500 mg/ml, Big CHAP can be present in a concentration of about 20 to about 360 mg/ml, Polysorbate 80 can be present in a concentration of about 1 to 36 mg/ml, the Pluronics can be present in concentrations of about 1 to about 150 mg/ml, and the other ingredients may be present in concentrations as set forth below.

[0097] The lyophilized formulations of SYN3 preferably contain a citrate buffering system. More preferably, the citrate buffering system can comprise at least one citric buffer, such as citric acid monohydrate USP or sodium citrate dihydrate USP. More preferably, the citrate buffering system comprises a combination of citric acid monohydrate USP and sodium citrate dihydrate USP. When used in combination, the amount of citric acid monohydrate USP can be present in a concentration of about 0.005 to about 2 mg/ml, more preferably 0.016 to about 1.35 mg/ml, preferably 0.016 to about 0.72 mg/ml, preferably about 0.005 to about 1.35, and the sodium citrate dihydrate USP can be present in a concentration of about 0.02 to about 5.37 mg/ml, preferably 0.05 to 3.00 mg/ml, preferably 0.05 to 2.31 mg/ml. Other suitable buffering systems that are suitable include, for example, phosphate, glycine, either in place of the citrate buffering system or in combination therewith, and varying combinations of all of the above.

[0098] The buffering system will provide a pH of the lyophilized formulation such that there is improved stability. Preferably, the pH will be in a range of about 5 to about 6. The admixture aqueous formulations of SYN3 are preferably buffered at about a pH of about 7 to about 8.5, preferably about 7.4, and SYN3 remains stable in the dehydrated powder for at least 3 months at 40° C.

[0099] The lyophilized formulations preferably contain glycine or mannitol as freeze-drying bulking agents. Other suitable freeze-drying bulking agents that may be used include, for example, lactose, recombinant gelatin, and methylcellulose. The freeze drying-bulking agent may be present in a concentration of from about 5 to 100 mg/ml when the agent is mannitol, and about 10 to 200 mg/ml when the agent is glycine.

[0100] The lyophilized formulations preferably contain ascorbic acid as an antioxidant. Other suitable antioxidants that may be used include, for example, citric acid. When ascorbic acids is the antioxidant, it may be present in a concentration of about 0.001 to about 0.6 mg/ml.

[0101] The compositions of this invention may additionally include, for example, a stabilizer, enhancer or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the recombinant adenoviral vector delivery system comprising the tumor suppressor gene. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, Hydroxypropyl-β-Cyclodextrin, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.

[0102] Other physiologically acceptable compounds include, for example, wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would know that the choice of pharmaceutically acceptable carrier, depends on the route of administration and the particular physiochemical characteristics of the recombinant adenoviral vector delivery system and the particular tumor suppressor gene contained therein. Examples of carriers, stabilizers or adjuvants can be found in Gennaro, Remington's: The Science and Practice of Pharmacy, 19th Ed. (Mack Publishing. Co., Easton, Pa. 1995), incorporated herein by reference.

[0103] The recombinant viral vector gene delivery system comprising a interferon gene formulated in a buffer comprising a delivery-enhancing agent may be delivered to any tissue or organ using any delivery method known to the ordinarily skilled artisan for example, intratumoral or intravesical administration. Cancerous tissues and organs include, for example, any tissue or organ having an epithelial membrane such as the gastrointestinal tract, the bladder, respiratory tract, and the lung. Examples include, but are not limited to, carcinoma of the bladder and upper respiratory tract, vulva, cervix, vagina or bronchi; local metastatic tumors of the peritoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma; carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose, larynx, esophagus, stomach, colon and rectum, gallbladder, or skin; or melanoma.

[0104] In some embodiments, the organ or tissue defines an interior space, sinus, ventricle, passage, volume, cavity, void or lumen lined with or containing the epithelial membrane to be contacted with the pharmaceutical composition of the invention. The surfaces or walls defining such serve to contain or retard or limit the bulk fluid movement or transfer of the pharmaceutical composition to another body portion and can allow a longer contact time of the epithelial membrane with the pharmaceutical composition. In some embodiments, the organ or tissue is a bladder (e.g., the urinary bladder) which has an epithelial membrane at the inner surface. In some embodiments, the organ is the stomach, uterus, the intestine, the esophagus, the mouth, the colon, the upper or lower GI tract, or the upper or lower respiratory tract. In some embodiments, the organ or tissue defines a space such as the peritoneal cavity and the epithelial surface is located on an epithelial surface capable of making fluidic contact with the space within the peritoneal cavity. In some embodiments, the tissue is a nervous system tissue and the lumen is a cerebral ventricle.

[0105] The subjects of the invention are mammals, including, but not limited to, mice, rats, primates, and particularly, humans. An exemplary organ for intravesical administration of the composition is the urinary bladder.

[0106] In one aspect, the invention provides a method for administering a recombinant adenovirus having a gene encoding an interferon polypeptide to an organ or tissue having an epithelial membrane, comprising administering a therapeutically effective amount of the therapeutic adenovirus before, during or after contacting the organ or tissue with a buffer comprising a detergent, including SYN3 or homologues and analogues thereof, in which the detergent is in an amount sufficient to enhance the delivery of the gene delivery system. Exemplary delivery enhancing compounds which are also homologs of SYN3 for use according to the invention include compounds of Formula II.

[0107] The delivery enhancing compounds may be used singly with the interferon gene delivery system, or in combination with each other, or in combination with another delivery-enhancing agent. The delivery enhancing agents, compositions and methods of the invention are useful for many applications that require delivery of interferon to a cell, tissue or organ. For instance, the formulations are useful in treating diseases and conditions, including cancers, cellular proliferative disorders, and chronic infections (e.g., infections with HCV, HBV, HIV, or CMV) as well as other conditions responsive to interferon therapy. “Cancer” generally pertains to a unrestrained proliferation of cells within a tissue or organ. It is intended to encompass conditions in which one or more cells is classified as cancerous, malignant, tumorous, precancerous, transformed, or as an adenoma or carcinoma, or any other synonym commonly used in the art for these conditions.

[0108] In some embodiments, the compositions of the invention comprise a “therapeutically effective” amount of a therapeutic agent in a buffer comprising a delivery-enhancing compound. “Therapeutically effective” as used herein refers to the prevention of, reduction of, or curing of a disease state such as cancer or viral infection or the symptoms thereof.

[0109] Therapeutically effective amounts of the pharmaceutical composition comprising the interferon gene in a recombinant viral vector delivery system formulated in a buffer comprising a delivery-enhancing agent can be administered in accord with the teaching of this invention. For example, therapeutically effective amounts of the interferon gene in the recombinant adenoviral vector delivery system formulated in a buffer containing a delivery-enhancing agent are in the range of about 10 8 particles/ml to 10 12 particles/ml, more typically about 10 8 particles/ml to 5×10 11 particles/ml, most typically 10 9 particles/ml to 10 11 particles/ml (PN/ml) or 10 10 particles/ml to 10 11 particles/ml (PN/ml). The foregoing dosages are generally administered as frequently as tolerated by the subject. It is common practice in the field of oncology to provide dosages at or near the maximum tolerated dose.

[0110] In one embodiment of the invention for the treatment of bladder cancer, the interferon gene delivery system is provided in conjunction with a delivery enhancing agent as part of a multi-course treatment regimen consisting of three weekly cycles of treatment, each cycle providing a single intravesical administration on day 1 or optionally on days 1 and 2 of each cycle. The typical manner of treatment of bladder cancer, a practitioner would insert a catheter in the urethra allowing the subject to void. A solution is prepared comprising the formulated delivery enhancing agent and the interferon gene delivery system and instilled into the bladder. The catheter is clamped so as to maintain the solution in the bladder for approximately one hour. It should be noted that longer exposure may be desirable to enhance the transduction of the epithelium but practical considerations generally limit such practice. For example one hour is generally the point at which the subject will feel the need to void. The effect of the delivery enhancing agent has been demonstrated to produce a prolonged effect such that the pre-exposure of the bladder to the delivery enhancing agent in advance of the instillation of the interferon gene delivery system will provide an enhanced delivery. This may be employed where compatibility between there are issues of potential compatibility between the gene delivery system and components of the formulation comprising the delivery enhancing agent. Following the desired exposure, the catheter clamp is removed from the catheter and the subject allowed to void. Interferon expression may be monitored by biopsy of bladder tissue or by assessing the level of secreted interferon in the urine. The foregoing procedure may be repeated as many times as tolerated by the patient to achieve a therapeutic effect. The therapeutic effect may be readily assessed by conventional means such as a cystoscope inserted into the bladder.

[0111] These formulations are administered in a volume corresponding to the size of the void to be filled or organ or tissue to be treated. For the human bladder, a suitable volume of administration will depend upon the age and size of the subject, and particularly of their bladder. Administered volumes may typically range from 50 to 500 ml, and more commonly from 50 to 250 ml. or from 100 to 200 ml. In some methods of administration, the amount to be administered can be economically conserved by inserting a balloon catheter into the vesical or bladder to be treated and inflating the balloon portion of the catheter so as to reduce the void volume to be occupied by the administered composition.

[0112] In addition, the above treatment regimens maybe supplemented or used in conjunction with other therapeutic treatment regimens to provide an enhanced therapeutic effect. The methods of the present invention may be supplemented for example by the conventional BCG and/or chemotherapeutic treatment regimens. It is standard of practice in the field of oncology to provide multiple treatment regimens to an individual to maximize the effect. The dose provided

[0113] In another aspect, the invention provides a kit having the delivery enhancing compound in a first container and the interferon gene delivery system in a second container. In one embodiment, the contents of the containers are combined in preparing a formulation comprising both the delivery enhancing agent and the gene delivery system and the combination formulation is administered to a subject. In another embodiment, the contents of the containers are administered as separate preparations to a patient. The delivery enhancing agent may be administered before, during or after administration of the gene delivery system. In an exemplary embodiment, they are administered concurrently or nearly concurrently. The formulations either one or both of the containers may be a lyophilized powder to be brought up in a pharmaceutically acceptable liquid carrier.

[0114] In another embodiment, the kit provides a first container containing SYN3 or a SYN3 homologue or analog capable of enhancing the delivery of a gene delivery system; and a second container containing the gene delivery system wherein the gene delivery system has an interferon gene operably linked to a genetic regulatory element modulating the expression of the interferon gene. In one further embodiment, the first container contains a lyophilized formulation of SYN3 or a SYN3 homologue or analog. In another embodiment, the second container contains a lyophilized formulation of the gene delivery system. In a further embodiment, both the first and second container hold a lyophilized formulation of their respective contents. In an exemplary further embodiment, the gene delivery system is a recombinant adenoviral vector having a gene encoding a polypeptide which is substantially identical to a human alpha-interferon polypeptide or is a human alpha-interferon polypeptide.

[0115] In some embodiments, the delivery-enhancing compound is included in the buffer in which the gene delivery system is formulated. The delivery-enhancing compound can be administered prior to the gene delivery system or concomitant with the gene delivery system. In some embodiments, the delivery-enhancing compound is provided with the gene delivery system by mixing a gene delivery system preparation with a delivery-enhancing compound formulation just prior to administration to the patient. In other embodiments, the delivery-enhancing compound and gene delivery system are provided in a single vial to the caregiver for administration.

[0116] In the case of a pharmaceutical composition comprising an interferon gene contained in a recombinant adenoviral vector delivery system formulated in a buffer which further comprises a delivery-enhancing agent, the pharmaceutical composition can be administered over time in the range of about 5 minutes to 3 hours, preferably about 10 minutes to 120 minutes, and most preferably about 15 minutes to 90 minutes. In another embodiment the delivery-enhancing agent may be administered prior to administration of the recombinant adenoviral vector delivery system containing the tumor suppressor gene. The prior administration of the delivery-enhancing agent may be in the range of about 30 seconds to 1 hour, preferably about 1 minute to 10 minutes, and most preferably about 1 minute to 5 minutes prior to administration of the adenoviral vector delivery system containing the tumor suppressor gene.

[0117] In some embodiments, the administrations according to the methods and compositions of the invention may be repeated over various intervals of time so as to increase the therapeutic effect. These intervals may be approximately daily, weekly, or monthly. The number of administrations will vary with the therapeutic regime and its duration. Generally, therapeutic administrations according to the invention may be given semi-weekly, weekly, biweekly, monthly, or bimonthly for up to 2 months, four months, six months or even longer. The frequency of administration may be tailored to the individual subject by monitoring the production or release of interferon in the subject or assessing the clinical responses of the patient (e.g., tumor reduction if the disease being treated is cancer).

[0118] Advantageously, the amount of interferon released into a bodily fluid (e.g., mucous, blood, urine) can be monitored to assess the efficacy of the administration and to adjust the dosage amount or administration frequency of the regime in order to maintain a predetermined level of interferon production as measured by the fluid level. Sensitive and accurate methods for monitoring interferon, such as ELISA methods, in bodily fluids are well known to one ordinary skill in the art. In an exemplary embodiment, the bodily fluid is the urine and the treated organ is the urinary bladder.

[0119] In another embodiment, the response of a subject to the therapeutic methods and compositions of the present invention is assessed by monitoring the size and number of tumor masses found in the treated organ or tissue. This monitoring may by measurement of tumor specific antigens or products in bodily fluids such as blood or urine or by direct imaging methods such as MRI or radioisotope imaging techniques.

[0120] Compositions formulated with the delivery-enhancing agents and gene delivery systems of the invention can be used to facilitate delivery of interferon genes of interest to cells, including in particular, the epithelial cells of organs and tissues. The therapeutic uses of interferon are known to one of ordinary skill in the art. For instance, compositions according to the invention are useful in the treatment of cancer. Cancerous tissues and organs suitable for treatment according to the inventive methods and compositions include any tissue or organ having an epithelial membrane such as the gastrointestinal tract, the bladder, respiratory tract, and the lung. Examples include but are not limited to carcinoma of the bladder and upper respiratory tract, vulva, cervix, vagina, uterus or bronchi; local metastatic tumors of the peritoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma; carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose, larynx, esophagus, stomach, colon and rectum, gallbladder, or skin; or melanoma.

[0121] The methods and compositions