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  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
  • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
  • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
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  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/02—Dextran; Derivatives thereof
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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  • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
  • C08L79/02—Polyamines
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10051—Methods of production or purification of viral material
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/95—Protection of vectors from inactivation by agents such as antibodies or enzymes, e.g. using polymers

Definitions

• Recombinant adenovirus is used extensively as a vector in gene therapy due to its ability to deliver genes into a wide variety of proliferating and non-proliferating cells.
• Systemic delivery of adenovirus faces several hurdles such as short blood half life (Morrissey et al, Toxicol Sci. 65: 266-275 (2002); Alemany et al., JGen Virol. 81: 2605- 2609 (2000)), elimination by the reticuloendothelial system (RES) (Ziegler et al., Hum Gene Ther. 13: 935-945 (2002); Tao et al, Mol Ther.
• RES reticuloendothelial system
• hydrophilic polymers such as PEG (O'Riordan et al, Hum Gene Ther. 10: 1349-1358 (1999); Croyle et al, Hum Gene Ther. 11: 1713-1722 (2000)) or pHPMA (Fisher et al, Gene Ther. 8: 341-348 (2001))
• PEG Olet al, Hum Gene Ther. 10: 1349-1358 (1999); Croyle et al, Hum Gene Ther. 11: 1713-1722 (2000)
• pHPMA Fisher et al, Gene Ther. 8: 341-348 (2001)
• Covalent attachment of adenovirus to an encapsulating polymer requires an additional purification step and subsequently lowers the chemical yield.
• infectivity of adenovirus is typically reduced when PEG is covalently attached (O'Riordan et al, Hum Gene Ther. 10: 1349-1358 (1999); Croyle et al, Hum Gene Ther. 11: 1713-1722 (2000)).
• the present invention addresses this and other problems.
• the present invention provides for noncovalent complexes of copolymers and adenoviruses.
• the copolymer which is a combination of a cationic polymer, such as PEI, polylysine, DEAE-Dextran, and derivatives thereof, and a nonionic polymer, such as PEG and derivatives thereof, can improve both delivery and transgene expression of the adenovirus in cells.
• the complex of the invention provides an easy-to-produce material that is therapeutically more effective than an unencapsulated adenovirus.
• the invention provides a complex comprising an adenovirus noncovalently complexed to a copolymer.
• the copolymer comprises a structure according to Formula I:
• m is an integer from 1 to 1 ,000.
• i is an integer from 2 to m and denotes the position of X'.
• the symbols X 1 , X 1 , and X m+1 are independently selected monomers, wherein (i) said monomers comprise an amine selected from secondary amines and tertiary amines; and (ii) at least one of said monomers comprise Q.
• Q is a structure selected from Formula Ila and Formula lib:
• the copolymers of the invention are also free of cross-polymerization, and, at physiological pH, at least one of the nitrogen atoms in the copolymer is positively charged.
• the copolymers of Formula I are involved in a method of preparing a noncovalently complexed adenovirus copolymer complex.
• the copolymers of Formula I are involved in a method of introducing an adenovirus into a cell.
• this method (a) an adenovirus is noncovalently contacted to a copolymer, and (b) the complex is contacted to a cell.
• the present invention provides a physiological formulation comprising: (a) a copolymer of Formula I; (b) an adenovirus, which forms a noncovalent complex with the adenovirus; and (c) a physiologically acceptable excipient.
• the present invention provides a kit comprising a copolymer of Formula I and an adenovirus, wherein the copolymer and adenovirus are noncovalently attached.
• Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
• the term encompasses the terms gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
• the term also encompasses synthetic, naturally occurring, and non-naturally occurring nucleotide analogs with modified backbone residues or linkages. These nucleotide analogs have similar binding properties as the reference nucleic acid, or are metabolized in a manner similar to the reference nucleotides.
• Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide- nucleic acids (PNAs).
• nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol Chem. 260:2605-2608 (1985); Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).
• contacting a cell refers to the intemalization of an adenovirus or complex of the invention into the cell. This term encompasses, for example, intravenous or oral administration of the virus or complex which results in its intemalization into the cell.
• adenovirus generally comprises a polynucleotide comprising all or a portion of an adenovirus genome.
• Adenovirus refers collectively to animal adenoviruses of the genus mastadenovirus including, but not limited to, human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera.
• human adenoviruses include the A-F subgenera 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 (Adl 1A and Ad I IP), 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.
• human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 Adl 1A and Ad I IP
• bovine adenoviruses useful in the invention include, but are not limited to, bovine adenovirus types 1,2,3,4,7, and 10.
• Canine adenoviruses include but are not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2.
• Equine adenoviruses of interest include, but are not limited to, equine types 1 and 2
• porcine adenoviruses of interest include, for example, porcine types 3 and 4.
• "Adenovirus” also refers collectively to recombinant adenoviruses, such as those produced from the deletion, insertion, or mutation of nucleic acids.
• the recombinant adenoviruses can also be produced from the linking of DNA from different serotypes or subgenera.
• noncovalent means the binding of substances via ionic bonding, electrostatic interactions, hydrogen bonding, hydrophilic-hydrophilic interactions, hydrophobic-hydrophobic interactions, van der Waals interactions and combinations thereof.
• polymer refers to natural and synthetic compounds of usually high molecular weight consisting of up to millions of repeated linked monomers. Each monomer is a relatively light and simple molecule.
• homopolymer refers to a polymer derived from a single type of monomer.
• copolymer refers to a polymer produced by the simultaneous polymerization of two or more dissimilar monomers.
• cross-polymerization refers to the covalent attachment of two or more polyalkylene imine moieties to opposing ends of a polyalkylene glycol molecule.
• substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH 2 O- is equivalent to -OCH 2 -.
• alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. Cj-C t o means one to ten carbons).
• saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like.
• An unsaturated alkyl group is one having one or more double bonds or triple bonds.
• alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
• alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.”
• Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl".
• alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
• an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
• a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
• alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
• heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
• the heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
• heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 - CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
• heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkyl enedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O) 2 R'- represents both -C(O) 2 R'- and -R'C(O) 2 -.
• cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively.
• a cycloalkyl or heterocycloalkyl include saturated and unsaturated ring linkages.
• a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
• Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like.
• heterocycloalkyl examples include, but are not limited to, l- ⁇ l,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.
• polyalkylene glycol refers to polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof.
• An exemplary embodiment of a polyalkylene glycol derivative is adipate dihydrazide-methyoxy- polyethylene glycol.
• Other exemplary embodiments are listed in Shearwater Corporation's catalog “Polyethylene Glycol and Derivatives for Biomedical Applications” (2001).
• aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together or linked covalently.
• heteroaryl refers to aryl groups (or rings) that contain from one 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-isoquinoly
• aryl when used in combination with other terms (e.g. , aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
• arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like).
• alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
• an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyl
• oxo as used herein means an oxygen that is double bonded to a carbon atom.
• R', R", R'" and R" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
• each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
• R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
• -NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
• alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
• Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer of from 0 to 3.
• two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer of from 1 to 4.
• One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
• two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR') s -X-(CR"R'")d-, where s and d are independently integers of from 0 to 3, and X is - O-, -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
• the substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C ⁇ -C 6 )alkyl.
• heteroatom is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
• the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
• the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
• the present invention provides compounds that are in a prodrug form.
• Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment.
• ring as used herein means an encircling arrangement of atoms optionally having heteroatoms within the arrangement.
• a ring includes aromatic and non-aromatic moieties such as substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.
• the number of atoms in a ring are typically defined by the number of members in the ring. For example, a "5- to 7- membered ring" means there are 5-7 atoms in the encircling arrangement. Each member is optionally a heteroatom.
• the term "5- to 7- membered ring” includes, for example pyridinyl, piperidinyl and thiazolyl rings. Rings are typically drawn with a single explicit substituent within parentheses having a subscript letter.
• the subscript letter typically represents a set of integers, such as 1-10.
• the integers represent the number of ring substituents wherein each substituent is optionally different. For example, for the substituent (R ! ) s , where s is 2, the ring may be substituted with a substituted or unsubstituted alkyl and a substituted or unsubstituted heteroalkyl.
• poly as used herein means at least 2.
• a polyvalent metal ion is a metal ion having a valency of at least 2.
• Moiety refers to the radical of a molecule that is attached to another structure.
• Fig. 2 Resource-Q anion exchange chromato grams of recombinant adenovirus (panel A) and adenovirus encapsulated with PEI-mPEG (panel B, encapsulation ratio C).
• the graphs show absorbance at 260 nm as a function of time.
• Chromatographic conditions were as follows: Flow rate: 1 mL/minute, buffer A: 50 mM Hepes, pH 7.5, buffer B: Buffer A + 1.5 M NaCl, gradient: 20% B to 40% B in 10 minutes.
• Fig. 3 In vitro infectivity and ⁇ -galactosidase expression of recombinant adenovirus and adenovirus encapsulated with PEI-mPEG.
• Cell lines used were T24 bladder carcinoma (white bars) and A549 lung carcinoma (black bars).
• Recombinant adenovirus expressing GFP (panel A) or /3-Galactosidase (panel B) was encapsulated with PEI-mPEG using three different ratios of polymer to virus particles (rAd(enc) A, rAd(enc) B, and rAd(enc) C) as described in Table 1.
• rAd(enc) A, rAd(enc) B, and rAd(enc) C three different ratios of polymer to virus particles (rAd(enc) A, rAd(enc) B, and rAd(enc) C) as described in Table 1.
• untreated cells UT
• Fig. 4 Quantitative PCR results for recombinant adenovirus DNA in livers (black bars), spleens (diagonally hatched bars), kidneys (horizontally hatched bars), and lungs (white bars) of BALB/c mice dosed intravenously with different doses of adenovirus or encapsulated adenovirus.
• Virus was encapsulated at the highest PEI-mPEG to virus particle ratio (C in Table 1). Groups were injected with 3xl0 10 , lxlO 10 , and 3xl0 9 virus particles per animal. Three animals per group were dosed.
• Fig. 5 ⁇ -galactosidase activity in livers (black bars), spleens (diagonally hatched bars), kidneys (horizontally hatched bars), and lungs (white bars) of BALB/c mice injected intravenously with different doses of adenovirus or encapsulated adenovirus.
• Virus was encapsulated at the highest PEI-mPEG to virus particle ratio (C in Table 1). Groups were injected with 3xl0 10 , lxlO 10 , and 3xl0 9 virus particles per animal. Three animals per group were dosed. Due to different background levels in the assay the limit of quantitation was 7.7 ng/g tissue for liver extracts and 0.1 ng/g for all other tissues.
• the present invention relates to a copolymer that noncovalently encapsulates viral particles, forming a complex.
• the complexes are easily made (e.g. by mixing virus and polymers) and can be used to express nucleic acids in cells.
• chemical modification of a complex biological material such as adenovirus is not required, thereby avoiding extensive characterization of possible reaction products.
• Introduction of nucleic acids into cells is useful for, e.g., therapeutic or diagnostic purposes (e.g. using a reporter gene).
• several experimental cancer therapies utilize various aspects of adenovirus or adenovirus vectors. See, for example, U.S. Pat. Nos.
• virus/polymer complexes of the invention can be used to transfer nucleic acids of interest into different cell types either in vitro, in vivo or ex vivo.
• the invention provides a complex comprising an adenovirus noncovalently complexed to a copolymer.
• the copolymer comprises a structure according to Formula I:
• m is an integer from 1 to 1,000.
• the symbol i is an integer from 2 to m and denotes the position of X 1 .
• the symbols X 1 , X 1 , and X m+1 are independently selected monomers, wherein (i) said monomers comprise an amine selected from secondary amines and tertiary amines; and (ii) at least one of said monomers comprise Q.
• Q is a structure selected from Formula Ila and Formula lib: (Ha); (Hb);
• the copolymers of the invention are also free of cross-polymerization, and, at physiological pH, at least one of the nitrogen atoms in the copolymer is positively charged.
• At least one of the monomers further comprises a chemical moiety selected from -NH 2 and -OH. If Q is selected from Formula Ila, then at least one Q is covalently linked to said monomers through an atom selected from nitrogen and oxygen. If Q is selected from Formula lib, then at least one Q is covalently linked to said monomers through a carbon atom.
• Q is Formula Ila
• h is 0, Z is O
• said monomers comprise a structure according to Formula III:
• c is an integer from 1 to 10.
• Q is Formula Ila, h is 0, Z is O
• said copolymer has: a) at least one monomer with a structure according to Formula III, wherein a is an integer from 1 to 10, and b is an integer from 1 to 10; and b) at least one monomer with a structure according to Formula IV; wherein c is an integer from 1 to 10.
• a is 2.
• b is 2.
• c is 2.
• a is 3.
• exemplary embodiment b is 3.
• c is 3.
• the monomer is ethylene imine.
• Q is Formula Ila
• h is 0, Z is O
• said monomers comprise a structure according to Formula V:
• the monomer is lysine.
• Q is Formula Ila
• h is 1
• Z is NH
• said monomers comprise a structure according to Formula VI:
• R 3 is a member selected from H, -(CH 2 CH2)NH(CH 2 CH 3 ) 2 , and - (CH 2 CH 2 )N(CH 2 CH 3 ) 2 CH 2 CH 2 NH(CH 2 CH 3 ) 2 .
• R 3 is H for about two of every three of said monomers, and for about one of every three of said monomers, R 3 is a member selected from -(CH 2 CH2)NH(CH 2 CH 3 )2, and - (CH 2 CH 2 )N(CH 2 CH 3 ) 2 CH 2 CH 2 NH(CH 2 CH 3 ) 2 .
• the monomer is DEAE-Dextran.
• Q is Formula lib
• said copolymer comprises: a) at lleeaasstt oonnee mmoonnoommeerr wwhhiicchh ccoommppririsseess aa ssttrruuccttuurree aaccccoorrddiinngg to Formula VI, wherein R is a member selected from H, -(CH 2 CH 2 )NH(CH 2 CH 3 ) 2 , and -(CH 2 CH 2 )N(CH 2 CH 3 ) 2 CH 2 CH 2 NH(CH 2 CH 3 )2; and b) at least one monomer which comprises a structure according to Formula VII:
• R 3 is H for about two of every three of said monomers, and for about one of every three of said monomers, R is a member selected from - (CH 2 CH 2 )NH(CH 2 CH 3 ) 2 , and -(CH 2 CH 2 )N(CH 2 CH 3 )2CH2CH 2 NH(CH 2 CH 3 ) 2 .
• the percentage of said monomers which comprise a structure according to Formula VII is between 5 and 25.
• R is H for about two of every three of said monomers, and for about one of every three of said monomers, R 3 is a member selected from -(CH 2 CH 2 )NH(CH 2 CH 3 ) 2 , and - (CH 2 CH 2 )N(CH 2 CH 3 ) 2 CH 2 CH 2 NH(CH 2 CH 3 ) 2 , and the percentage of said monomers which comprise a structure according to Formula VII is between 5 and 25.
• the non-ionic polymer contains a polyalkylene glycol moiety, which is covalently attached to some of the monomers. In certain embodiments, this covalent attachment results in the formation of a secondary or tertiary amine, an amide, a dihydrazide, an ester, a urea, an isourea, a carbamate, or an urethane.
• polyalkylene glycols include polyethylene glycol (PEG) and its derivatives.
• a non-ionic polymer such as polyethylene glycol (PEG)
• a cationic polymer such as PEI, polylysine, DEAE-Dextran and variants thereof
• PEG functions to increase transfection efficiency.
• R 1 comprises a structure according to Formula VIII:
• R 2 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted 3- to 7- membered cycloalkyl, substituted or unsubstituted 5- to 7- membered heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
• p is 2.
• the copolymers of the invention have different levels of non-ionic polymer substitution on the monomers of the cationic polymer. For example, 15% of the ethylene imine monomers in the cationic polymer PEI are substituted with the non-ionic polymer PEG. In some cases, the level of non-ionic polymer substitution is between 10% and 20%. In other cases, the level of non-ionic polymer substitution is between 10% and 30%. In other cases, the level of non-ionic polymer substitution is between 15% and 25%. In other cases, the level of non-ionic polymer substitution is about 20%. In other cases, the level of non-ionic polymer substitution is between 10% and 40%.
• the percentage of monomers which are substituted with Q is at least 10% and Q has a structure according to Formula IX:
• R 2 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted 3- to 7- membered cycloalkyl, substituted or unsubstituted 5- to 7- membered heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
• p is 2.
• the percentage of monomers which are substituted with Q is from 15 to 30.
• the percentage of monomers which are substituted with Q is from 17 to 22. [0063]
• the percentage of monomers which are substituted with Q is at least 10 and Q has a structure according to Formula X:
• the symbol n is an integer from 2 to 2,000.
• the symbol p is an integer from 1 to 8.
• the ssyymmbbooll ff i iss an integer from 0 to 1.
• the symbol R 4 has a structure according to Formula XI when f is 1 :
• N' is covalently attached to N*, and g is an integer from 1 to 9.
• R 2 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted 3- to 7- membered cycloalkyl, substituted or unsubstituted 5- to 7- membered heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
• f is 0.
• f is 1.
• g is 4.
• the percentage of monomers which are substituted with Q is from 15 to 30.
• the percentage of monomers which are substituted with Q is from 17 to 22.
• the percentage of monomers which are substituted with Q is at least 10 and Q has a structure according to Formula XII:
• R 2 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted 3- to 7- membered cycloalkyl, substituted or unsubstituted 5- to 7- membered heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
• the percentage of monomers which are substituted with Q is from 15 to 30.
• the percentage of monomers which are substituted with Q is from 17 to 22.
• the complex has a diameter between about 20 nm and about 300 nm. In another exemplary embodiment, the complex has a diameter between about 80 nm and about 150 nm.
• R is a member selected from substituted and unsubstituted alkyl, substituted and unsubstituted aryl, folate, transferrin, galactose, glucose, antibodies, antibody fragments, and peptides.
• R 2 is methyl.
• p is 2.
• p is 2, a is 2, b is 2, and c is 2.
• the ratio of the copolymer to the adenovirus is between 3,500:1 and 30,000:1. In another exemplary embodiment, the ratio of the copolymer to the adenovirus is between 3,600:1 and 20,000:1. In still another exemplary embodiment, the ratio of the copolymer to the adenovirus is between 10,000:1 and 30,000:1.
• the copolymers of the invention can possess a range of physical dimensions.
• some of the copolymers of the invention have an average molecular weight of between about 100 and about 300 kilodaltons (kDa).
• the average molecular weight is between 125 and 250 kDa.
• the average molecular weight is between about 150 and about 170 kDa.
• the length of the copolymer is not critical, as long as the complex it forms with the adenovirus is substantially electroneutral.
• the symbol m is an integer from 10 to 900.
• the symbol m is an integer from 50 to 600.
• the symbol m is an integer from 75 to 300.
• the copolymers in their most general form, are formed by reacting a cationic polymer with a non-ionic polymer that, for the exception of one end, is capped with non- reactive groups.
• the copolymer PEI-mPEG is formed by the reaction of a cationic polymer PEI with a non-ionic polymer PEG which is capped at one end with a methoxy group and possesses a succinimidyl propionate group at the other end.
• a nonionic, hydrophilic polymer such as polyalkylene glycol is covalently attached to a cationic polymer in this invention.
• Suitable polyalkylene glycols are commercially available from many sources, including polypropylene glycol and poly(l,2 butylene glycol) from Aldrich Chemical Company, and polyethylene glycol and its derivatives from Nektar Therapeutics.
• the polyalkylene glycol subunit is covalently attached to the cationic polymer through a group such as a secondary or tertiary amine, an amide, a dihydrazide, an ester, a urea, an isourea, a carbamate, an urethane, or combinations thereof.
• the number of polyalkylene glycol subunits (n) can be, e.g., between 2-2,000. In some compounds of the invention, the number of subunits is between 45-1,200. In other compounds of the invention, the number of subunits is between 250-1,000.
• polyalkylene glycol can be capped, e.g. , with a group forming an ether linkage, such as an alkoxy group.
• a group forming an ether linkage such as an alkoxy group.
• monomethoxypolyethylene glycol (mPEG) is used as a capping group.
• capping groups are substituted and unsubstituted alkyl, substituted and unsubstituted aryl, folate, transferrin, galactose, glucose, antibodies, antibody fragments, and peptides.
• capping groups for PEG can be found in the Nektar Therapeutics (formerly Shearwater Polymers) (Birmingham, AL) 2001 catalog (available on the Internet (World Wide Web) at nektar.com), which is herein incorporated by reference.
• the non-ionic polymer before coupling to the cationic polymer, is chemically activated.
• activated PEG in this example, mPEG
• mPEG include cyanuric chloride mPEG, succinimidyl succinate mPEG, tresyl- mPEG, and succinimidyl propionate mPEG (mPEG-SPA).
• activated PEG groups can also be found in the Nektar Therapeutics catalog mentioned above.
• the cationic polymers of the invention comprise the symbols X 1 , X 1 , and X m+1 which represent positively charged monomers. These positively charged monomers form a cationic polymer. These positively charged monomers contain secondary or tertiary amines, and can contain primary amine or alcohol functionalities as well.
• the cationic polymers of the invention can have an average molecular weight of between about 800 and about 800,000 daltons. In some embodiments, the average molecular weight is between 2,000 and 100,000 daltons. In other embodiments, the average molecular weight is between about 15,000 and about 50,000 daltons. Additionally, the length of the cationic polymer, which can be represented by m, can vary between 1 and 1000. In some embodiments, m is a whole number between 10 and 900. In another embodiment, m is a whole number between 50 and 600. In still another embodiment, m is a whole number between 75 and 300.
• the cationic polymers are polyalkylene imines, polylysine, DEAE-Dextran, and DEAE-Dextran variants.
• the cationic polymer is a polyalkylene imine.
• polyalkylene imine contains the following monomer:
• polyalkylene imine contains the following monomer: in which, c is an integer between 1-10. In some cases, c is 2. In other cases, c is 3. In still other cases, polyalkylene imine is a mixture of the monomers listed above. In yet another case, polyalkylene imine is polyethylene imine (PEI).
• PEI polyethylene imine
• polyalkylene imine contains monomers of a structure according to Scheme I, in which the ratio of x to y is between 1:1 and 5:1. The ratio can also be, e.g., 2:1 or even 3:1.
• Suitable polyalkylene imine compounds are commercially available from many sources, including polyethylenimine from Aldrich Chemical Company, polyethylenimine from Polysciences, and POLYMIN poly(ethylenimine) and LUPASOLTM poly(ethylenimine) available from BASF Corporation.
• polylysine is commercially available from many sources, including Sigma Chemical Company. Polylysine is composed of monomers having the following structure:
• Polylysine-PEG copolymers are produced by the method of Scheme 2.
• polylysine is reacted with a methoxy-capped, succinimidyl propionate-activated PEG in an aqueous solution containing a borate buffer in order to produce a polylysine-mPEG copolymer.
• Cationic Polymers DEAE-Dextran
• DEAE-Dextran hydrochloride is commercially available from many sources, including Sigma Chemical Company.
• DEAE-Dextran can contain a mixture of monomers such as the ones listed below:
• a saccharide is reacted with cyanogen bromide in a 60% acetone:30% water:10% triethylamine solvent at -15°C in order to produce an activated saccharide nitrile.
• a DEAE-Dextran-mPEG isourea is formed.
• the ratio of unsubstituted glucose subunits to DEAE-substituted glucose (including single and multiple DEAE- substitutions) subunits is between 1 :1 and 5:1.
• the ratio can also be, e.g., 2:1 or even 3:1.
• DEAE-Dextran-PEG copolymers with an adipate dihydrazide linker are produced by the method of Scheme 4.
• a saccharide is reacted with cyanogen bromide in a 60% acetone:30% water: 10% triethylamine solvent at -15°C in order to produce an activated saccharide nitrile.
• the activated polymer is first reacted with adipic acid dihydrazide, and subsequently reacted with an aldehyde functionalized mPEG in the presence of sodium cyanoborohydride to furnish DEAE-Dextran-PEG copolymers with an adipate dihydrazide linker.
• the ratio of unsubstituted glucose subunits to DEAE-substituted glucose (including single and multiple DEAE-substitutions) subunits is between 1:1 and 5:1.
• the ratio can also be, e.g., 2:1 or even 3:1.
• DEAE-Dextran-PEG copolymer variants are produced.
• DEAE-Dextran variants can contain the monomers listed above.
• DEAE- Dextran-PEG copolymer variants are produced by the method of Scheme 5.
• Viruses are highly efficient at nucleic acid delivery to specific cell types, while often avoiding detection by the infected hosts' immune system. These features make certain viruses attractive candidates as gene-delivery vehicles for use in gene therapies. Retro virus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus are examples of commonly used viruses in gene therapies. Each of the aforementioned viruses has its advantages and limitations, and can therefore be selected according to its suitability for a given gene therapy.
• Adenoviruses as vectors for the delivery of foreign DNA are used extensively as tools of modern molecular biology. Adenoviral replication does not require the recipient host cell to be dividing, in contrast to most retrovirus vectors. Adenoviruses can be designed to enter mammalian cells and express proteins but can be otherwise defective for production of infectious progeny virus.
• Adenovirus vectors can be prepared to be either replication competent or conditionally defective in a variety of genes required for a productive infection. For instance, from experience with adenovirus-SV40 recombinants, it was learned that the entire adenovirus E3 region could be deleted without a major change in viral growth in tissue- cultured cells. This region can be substituted with foreign DNA. The resulting adenovirus can be grown in any cell line permissive for wild-type adenovirus infection. E3-substituted adenoviruses have been used primarily for the insertion of genes to produce proteins for immunization.
• viruses engineered as tools for gene therapy are generally designed to be defective for replication. Most of the foreign DNA for the latter vectors replace the deleted El A and EIB regions.
• the constructs are made by a variety of protocols but include plasmids into which the foreign DNA is inserted, flanked by adenovirus sequences.
• adenoviruses can be used to facilitate the insertion of foreign DNAs into cells.
• the mechanisms of viral attachment, processing through the endosomes, and eventual delivery of DNA to the nucleus can be used to co-internalize foreign DNA external to the adenovirus particle.
• DNA complexed with polylysine can be joined to adenovirus particles and co-internalized. See, e.g., Cottam, J Virol 67: 3777-3785 (1993), Wagner, PNAS 88: 4255-5259 (1991).
• the complex enters cells presumably by attachment of the fiber to its putative receptor, but there have been modifications of these techniques to include ligands, like transferrin, to facilitate entry into cells with the transferrin receptor. See, e.g., Wagner, EN4S 89: 6099-6103 (1992). It is unnecessary to link the virus and the external D ⁇ A for efficient entry of the D ⁇ A into cells in culture. See, e.g., Yoshimura, JE/o/ Chem 268: 2300-2303 (1993).
• the invention provides complexes comprising an adenovirus and a copolymer.
• These copolymers can be of a kind described in part A above.
• the adenovirus and copolymer are noncovalently complexed to one another.
• the complex of the invention can possess a range of physical dimensions.
• the amount of the copolymer versus the amount of adenovirus in the complex can vary.
• the ratio of the copolymer to the adenovirus is between 3,500:1 and 30,000:1. In other embodiments, the ratio of the copolymer to the adenovirus is between 3,600:1 and 20,000:1. In still other embodiments, the ratio of the copolymer to the adenovirus is between 10,000:1 and 30,000:1.
• the size of the complex can vary. In some embodiments, the complex is between about 20 nm and about 300 nm. In other embodiments, the complex is between about 80 nm and about 150 nm.
• the copolymers of the invention are simply contacted to, or mixed with, an adenovirus (See Example 2). Complex formation occurs almost instantaneously, and no further purification is needed. The complexes of the invention can then be used for contacting a cell. Contacting the complexes of the invention to cells results in the introduction of the virus or viral nucleic acids into the cell.
• Non-covalent encapsulation of adenovirus with cationic PEI-PEG copolymer results in a complete masking of virus surface charges while increasing virus infection and transgene expression in vitro and in vivo. This surface modification was achieved through a simple mixing process, with no further processing steps. PEI-PEG can be added to any purified virus preparation as a formulant, thereby allowing its use with any previously generated viral vectors.
• the cells are contacted with the complex in vitro. In some embodiments, the cells are contacted with the complex in vivo.
• the invention provides a method of preparing a noncovalently complexed adenovirus copolymer complex.
• the copolymer as described in this section, is contacted with an adenovirus.
• the invention provides a method of introducing an adenovirus into a cell.
• a copolymer is noncovalently contacted to an adenovirus, which forms the complex. This complex is then contacted to a cell.
• the invention provides a physiological formulation comprising: (a) a copolymer; (b) an adenovirus, which forms a noncovalent complex with the adenovirus; and (c) a physiologically acceptable excipient.
• the copolymer in this physiological formulation comprises a structure according to Formula I wherein m is an integer from 1 to 1,000.
• the symbol i is an integer from 2 to m and denotes the position of X 1 .
• the symbols X 1 , X 1 , and X m+1 are independently selected monomers, wherein (i) the monomers comprise an amine selected from secondary amines and tertiary amines; and (ii) at least one of the monomers comprise Q.
• Q is a structure selected from Formula Ila and Formula lib, wherein Z is selected from the group consisting of O and NH.
• the symbol h is an integer from 0 to 1.
• the symbol R comprises a polyalkylene glycol moiety.
• the copolymers of the invention are also free of cross-polymerization, and at physiological pH, at least one of the nitrogen atoms in the copolymer is positively charged.
• Physiologically acceptable excipients include, e.g., materials such as carriers, water, pH-adjusting or buffering agents, preservatives, stabilizers or other ingredients.
• a physiologically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the recombinant adenoviral vector delivery system.
• a physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, hydroxypropyl-jS-cyclodextrin, antioxidants, such as ascorbic acid or glutathione, chelating agents, water, low molecular weight proteins or other stabilizers or excipients.
• 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.
• physiologically acceptable carrier depends on the route of administration and the particular physiochemical characteristics of the recombinant adenoviral vector delivery system. Examples of carriers, stabilizers or adjuvants can be found in Gennaro, REMINGTON'S: THE SCIENCE AND PRACTICE OF PHARMACY, 19th ed. (1995) Mack Publishing Co., Easton, PA, incorporated herein by reference.
• the compounds of the present invention can be prepared and administered in a wide variety of oral, parenteral and topical dosage forms.
• the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally.
• the compounds described herein can be administered by inhalation, for example, intranasally.
• the compounds of the present invention can be administered transdermally.
• the present invention also provides physiological compositions comprising a physiologically acceptable excipient and a complex composed of a copolymer from the "Copolymer" section provided above, and a virus from the "Virus" section provided above.
• an effective amount of the complex is administered to a patient as a composition in a pharmaceutically acceptable excipient, including, but not limited to, saline solutions, suitable buffers, preservatives, stabilizers, and may be administered in conjunction with suitable agents such as antiemetics.
• An effective amount is an amount sufficient to effect beneficial or desired results, including clinical results.
• An effective amount can be administered in one or more administrations.
• an effective amount of a complex is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state or to diagnose a particular tissue or disease state.
• Site-specific injections of vector may include, for example, intraperitoneal, intrapleural, intrathecal, intra-arterial, intra-ocular, intra-tumor injections or topical application. These methods are easily accommodated in treatments using the combination of the complex and other agents, as appropriate.
• the complexes may be delivered to the target cell in a variety of ways, including, but not limited to, liposomes, general transfection methods that are well known in the art (such as calcium phosphate precipitation or electroporation), direct injection, and intravenous infusion.
• the means of delivery will depend in large part on the particular complex (including its form) as well as the type and location of the target cells (i.e., whether the cells are in vitro or in vivo).
• the viral dosage amounts for humans can range from 1 X 10 6 and 1 X 10 14 .
• Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
• liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
• the invention provides a kit comprising a copolymer and an adenovirus, wherein the copolymer and adenovirus are noncovalently attached.
• the copolymer of the kit comprises a structure according to Formula I wherein m is an integer from 1 to 1,000.
• the symbol i is an integer from 2 to m and denotes the position of X 1 .
• the symbols X 1 , X 1 , and X m+1 are independently selected monomers, wherein (i) said monomers comprise an amine selected from secondary amines and tertiary amines; and (ii) at least one of said monomers comprise Q.
• Q is a structure selected from Formula Ila and Formula lib, wherein Z is selected from the group consisting of O and NH.
• the symbol h is an integer from 0 to 1.
• the symbol R 1 comprises a polyalkylene glycol moiety.
• the copolymers of the invention are also free of cross-polymerization, and at physiological pH, at least one of the nitrogen atoms in said copolymer is positively charged.
• kits for the preparation of viruses for the infection of cells e.g., for gene therapy.
• the kits comprise, for example, the copolymers of the invention.
• the kits can contain a virus such as an adenovirus in a separate container.
• the kit can optionally contain written instructions describing how to use the invention.
• Other materials useful in the performance of the assays can also be included in the kits, including test tubes, transfer pipettes, and the like.
• the kits of the present invention can contain materials sufficient for one assay, or can contain sufficient materials for multiple assays.
• temperatures are given in degrees Celsius (°C); operations were carried out at room or ambient temperature, "rt,” or “RT,” (typically a range of from about 18-25 °C); evaporation of solvent was carried out using a rotary evaporator under reduced pressure (typically, 4.5-30 mm Hg) with a bath temperature of up to 60 °C; the course of reactions was typically followed by TLC and reaction times are provided for illustration only; yields are provided for illustration only; and the following conventional abbreviations are also used: L (liter(s)), mL (milliliters), mmol (millimoles), g (grams), mg (milligrams), min (minutes), and h (hours).
• GFP green fluorescent protein
• GFCB CMV immediate early promoter
• zeta potential was measured on a DELSA 440 SX (Coulter, Miami, FL). Coated viral particles were analyzed by anion-exchange chromatography on a Resource Q column (Amersham Biosciences, Piscataway, NJ) (Shabram, P.W., et al. Hum Gene Ther. 8:453-465 (1997)). Flow cytometry was conducted on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA); forward scatter (FSC), side scatter (SSC) and FL-1 parameters were collected for a total of 50,000 cells. Total protein content of cell and tissue lysates was determined by the BCA method (Pierce, Rockford, IL) relative to a bovine serum albumin (BSA) standard.
• BSA bovine serum albumin
• PEI 25 kDa was purchased from Aldrich (Milwaukee, WI), and methoxy-PEG- SPA (5 kDa) was obtained from Shearwater (Huntsville, AL). Analysis of the starting materials by reverse phase HPLC (linear gradient from 100% of 0.1% TFA in water to 100% of 0.1% TFA in acetonitrile, 20 minutes, flow rate: 0.4 mL per minute) showed a single peak at 10 minutes for PEI and 15 minutes for mPEG.
• PEG grafted polymer was synthesized by reaction of PEI with mPEG-SPA as shown in Scheme 1.
• a 1M PEI solution was prepared by dissolving 2.155 grams of PEI (25 kDa) in 10 mL of water, adjusting the pH to 7.0 with IN HCl, and adjusting the volume to 50 mL with water.
• To 153.9 milligrams of mPEG-SPA (3.0 x 10 "5 mol) was added 400 ⁇ L of PEI stock solution (1.27 x 10 "4 mol primary amines) diluted in 800 ⁇ L of borate buffer (200mM borate, 150 mM NaCl, ImM EDTA, pH 8.4). The reaction was allowed to progress for 1 hour at room temperature with shaking.
• reaction mixture was purified by size exclusion chromatography. Fractions were collected; those containing PEI-mPEG copolymer were pooled and the pool analyzed by reverse phase chromatography.
• the ratio of PEG to PEI in the copolymer was determined by comparing the 1H- NMR peak areas of the -CH 2 -CH 2 -O- protons in PEG and the -CH 2 CH 2 N- protons of PEI. Based on this data, the PEI-mPEG copolymer contained an average of 27.2 PEG chains per PEI molecule. This corresponds to a degree of modification of 18.8% of primary amines and an average molecular weight of 161 kDa.
• Recombinant adenovirus was treated with PEI-mPEG copolymer at various ratios by (1) diluting adenovirus into phosphate buffered saline (PBS) or vPBS (1 x PBS, 3 % w/v sucrose, 2 mM magnesium chloride, pH 7.5) to a final concentration of 1 x 10 11 particles per mL, (2) adding PEI-mPEG stock, and (3) mixing by pipetting. Complex formation was allowed to occur for 15 minutes at room temperature before use. No further purification was needed.
• PBS phosphate buffered saline
• vPBS vPBS (1 x PBS, 3 % w/v sucrose, 2 mM magnesium chloride, pH 7.5
• Complex formation was allowed to occur for 15 minutes at room temperature before use. No further purification was needed.
• Particle size was determined by dynamic light scattering. The reported hydrodynamic diameter was determined by measuring the Brownian motion of adenovirus particles in an aqueous buffer. Addition of PEI-mPEG copolymer resulted in a reduction of the surface charge in a dose-dependent manner and approached neutrality at the higher polymer concentrations. Since the surface charges were masked, it was surmised that the adenovirus particle was encapsulated with PEI-mPEG polymer. This binding occurred through charge interaction between the polycationic PEI backbone and the negatively charged virus surface.
• PEI-mPEG treated adenovirus was incubated for one hour in 50% v/v fetal bovine serum (FBS) in PBS at room temperature. The sample was subsequently tested by Resource Q HPLC analysis. No adenovirus peak was observed at the "typical" adenovirus retention time, indicating that the polymer was not displaced from the rAd by FBS. The retention time of untreated control adenovirus incubated in 50% v/v FBS did not change.
• FBS v/v fetal bovine serum
• the virus samples were centrifuged in a Beckman SW41 Ti rotor for 1 hour at ⁇ 154,000 x g, 8°C.
• the virus band from each tube was collected and mixed with 1.30 gm/ml CsCl in 10 m Tris-HCl, pH 8.0. Centrifugation was continued overnight in a VTi 65.3 rotor at ⁇ 199,000 x g, 8°C.
• Each virus band was collected, dialyzed in 3% sucrose in 2 m MgCl and lx PBS, pH 7.4 using Spectra/Por membrane with MWCO 50,000 (Spectrum Medical Industries, Inc., Houston, Texas). Dialyzed virus was stored frozen at - 80°C.
• A549 cells human epithelial lung carcinoma cells, express the Coxackie Adenovirus Receptor (CAR) and therefore are easy to infect with adenovirus.
• A549 cells were maintained in DMEM supplemented with 10% FBS at 37°C in a 7% CO 2 incubator.
• T24 cells are CAR negative and much less infectible than A549 cells.
• T24 human epithelial bladder carcinoma cells
• FITC red blood cells
• DME low glucose fetal calf serum
• FBS fetal bovine serum
• T24 human epithelial bladder carcinoma cells
• Cultures were grown in T-225 cm 2 tissue culture flasks until approximately 80% confluent, detached using 0.25% trypsin and seeded at 5 x 10 5 cells per well in 6-well plates. Cells were maintained overnight (37°C, 7% CO 2 ) before infection with adenovirus or PEI-mPEG encapsulated adenovirus.
• Infectivity was determined using a recombinant adenovirus expressing the green fluorescent protein (GFP).
• the transduction frequency i.e. percentage of GFP positive (infected) cells, was determined by flow cytometry.
• Transduction frequency was determined by dividing the number of FITC (FL-1) positive cells by the total number of analyzed cells.
• Transgene expression in vitro was evaluated in the same two cell lines using recombinant adenovirus expressing ⁇ -galactosidase (Figure 3B).
• Cells were infected with BGCG or BGCG encapsulated with PEI-mPEG in the same manner as described for the infectivity assay.
• Cells were lysed and transgene expression analyzed using a chemiluminescent ⁇ -galactosidase reporter gene assay kit (Roche, Mannheim, Germany). Briefly, 24 h post infection cells were washed with PBS and lysed with a mild detergent for 30 minutes at room temperature.
• ⁇ -galactosidase concentration was determined relative to a ⁇ -galactosidase standard (provided with kit) and normalized with the total protein content of the lysates.
• Biodistribution and transgene expression was evaluated after intravenous injection of the recombinant adenovirus or encapsulated adenovirus into the tail veins of BALB/c mice. Organs/tissues were harvested three days post injection and analyzed for the presence of adenoviral DNA by quantitative PCR and for ⁇ -galactosidase enzyme activity using a chemiluminescent ⁇ -gal reporter gene assay kit.
• mice Female BALB/c mice were slowly (300 ⁇ l in 20 seconds) injected with BGCG or BGCG encapsulated with PEI-mPEG into the tail vein. Three days after dosing, animals were sacrificed and the livers and tissues resected. The tissues were immediately placed in OCT and snapped frozen at -70 °C. 6 ⁇ m sections were cut and then stained with X-gal. Sections of each tissue were snap frozen in liquid nitrogen. There were 3 animals per dosing group.
• ⁇ -galactosidase enzyme activity in tissue samples Tissue samples were weighed into lysing matrix tubes (Q Biogene, Carlsbad, CA) and 0.2 mL of detergent lysis buffer with protease inhibitor cocktail (provided with ⁇ -Gal kit) per 100 mg of tissue was added. Tissue samples were lysed in a FastPrep tissue homogenizer (Q Biogene, Carlsbad, CA). After undergoing 3 freeze-thaw cycles, lysates were incubated at 50°C for 1 hour to inactivate endogenous ⁇ -galactosidase activity. Lysates were clarified by centrifugation at 14,000 x g for 10 minutes. Supernatant was removed and diluted aliquots analyzed for ⁇ -Gal enzyme activity using a chemiluminescent ⁇ -gal reporter assay kit (Roche, Mannheim, Germany).
• Primer and probe sequences used for PCR were as follows: BGCG forward primer, 5'-AACGGTACTCCGCCACC-3'; BGCG reverse, 5'-ACTGGTTAGACGCCTTTCTCGA- 3'; and BGCG probe, FAM-TCCGCATCGACCGGATCGG-TAMRA; murine GAPDH forward, 5'GAAGGTGAAGGTCGAGTC-3 ⁇ and GABDH reverse 5'-GAAGATGGTGATGGGATTTC-3'.
• the probe was FAM- CAAGCTTCCCGTTCTCAGCC-TAMRA.
• the PCR thermal profile was 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 62°C for 1 minute.
• diluted viral DNA isolated from BGCG was used as a viral DNA standard. Data from Q-PCR was expressed as viral copies/mg of tissue. The detection limit was approximately 10 copies/mg of tissue.
• Transgene expression was analyzed by measuring ⁇ -galactosidase enzyme activity in extracts prepared from aliquots of the harvested tissues (Figure 5).
• ⁇ -gal activity was at background levels for all doses except at 3xl0 10 particles.
• One exception was the liver sample from one animal dosed at 3xl0 9 particles, which showed a doubling of background expression (14.6 ng/g tissue).
• animals dosed with PEI- mPEG encapsulated adenovirus showed dose-dependent expression throughout the dosing range.
• expression was highest in liver, followed by spleen, and lowest in kidneys and lung.
• Transgene expression in the liver was visualized by histological analysis of tissue cryo-sections. This analysis was used as a means to assess transduction frequency in vivo. Some mice were injected with various doses of BGCG while others were injected with various doses of PEI-mPEG-BGCG. While the number of transgene expressing cells was approximately equal for animals dosed at 3xl0 10 particles, increased expression was observed for animals treated with PEI-mPEG encapsulated virus at the other dose levels. Very few positive cells were detected in the livers of animals dosed with non-encapsulated adenovirus, while a significant number of positive cells were detected when PEI-mPEG encapsulated rAd was injected.
• Intravenous injection of recombinant adenovirus results in a highly non-linear dose response in various immune competent mouse strains. This was observed in various organs but mostly reported in the liver, the organ with the highest expression after i.v. injection (Ziegler et al, Hum Gene Ther. 13: 935-945 (2002), Tao et al, Mol Ther. 3: 28-35 (2001)). This effect is attributed to the existence of a "biological filter" which manifests itself by preferential uptake of adenovirus by Kupffer cells as well as other parts of the reticuloendothelial system (Tao et al, Mol Ther. 3:28-35 (2001)).
• Encapsulation of adenovirus with PEI-PEG copolymer provides a practical strategy to reduce the interaction of recombinant adenovirus with the RES and improve the therapeutic potential of the vector system.
• viral DNA levels were higher in all tissues for encapsulated adenovirus when compared to non-encapsulated virus injected intravenously at the same dose. This suggests encapsulation with PEI-PEG protected the adenovirus from clearance.
• expression in all analyzed tissues was increased with the largest increases seen in the liver.

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