WO2001083698A2 - Compositions and methods for inducing activation of dendritic cells - Google Patents

Compositions and methods for inducing activation of dendritic cells Download PDF

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Publication number
WO2001083698A2
WO2001083698A2 PCT/US2001/013921 US0113921W WO0183698A2 WO 2001083698 A2 WO2001083698 A2 WO 2001083698A2 US 0113921 W US0113921 W US 0113921W WO 0183698 A2 WO0183698 A2 WO 0183698A2
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composition
block copolymer
pluronic
polynucleotide
copolymer
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PCT/US2001/013921
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English (en)
French (fr)
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WO2001083698A3 (en
Inventor
Alexander V. Kabanov
Pierre Lemieux
Nadia Guerin
Valery Alakhov
Serguie Vinogradov
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Supratek Pharma, Inc.
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Priority to EP01941463A priority Critical patent/EP1283727A4/en
Priority to AU2001274815A priority patent/AU2001274815A1/en
Priority to JP2001580308A priority patent/JP2004509838A/ja
Priority to CA002407700A priority patent/CA2407700A1/en
Publication of WO2001083698A2 publication Critical patent/WO2001083698A2/en
Publication of WO2001083698A3 publication Critical patent/WO2001083698A3/en

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    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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Definitions

  • compositions and methods for activation of dendritic cells by administering compositions comprising polynucleotides, such as viruses, RNA, DNA, or derivatives thereof, and at least one block copolymer of an alkyether.
  • antigens are unable to promote an effective immune response against the pathogen.
  • certain antigens may elicit only a certain type of immune response, for example, a ce ( l- mediated or a humoral response.
  • Adjuvants are substances that enhance, augment or potentiate an immune response, and can in some instances, be used to promote one type of immune response over another.
  • numerous vaccine adjuvants are known, aluminum salt is the only adjuvant widely used in humans, not, however, without any safety concern.
  • DC Dendritic cells
  • APC antigen presenting cells
  • DC express constitutively, or after maturation, several molecules that mediate physical interaction with and deliver activation signals to responding T cells. These include class I and class II MHC molecules.
  • CDSO B7-1
  • CD86 B7-2
  • CD 40 CD 40;
  • DCs Dendritic cells
  • Other cells are also known to be able to present antigens such as macrophages and B-cells.
  • macrophages cannot take up soluble antigens efficiently, while immature dendritic cells can take up large amount of antigen from extracellular fluid by macropinocytosis.
  • B-cells are uniquely adapted to bind specific soluble molecules through their cell-surface immunoglobulin.
  • B-cells internalize the soluble antigen bound by their immunoglobulin receptors and then display peptide fragments of these antigens as peptides: MHC class II complexes.
  • MHC class II complexes The problem with B-cells is that they do not constitutively express co-stimulatory activity. Although B-cells efficiently present soluble proteins, they are unlikely to initiate a potent CTL response in the absence of co-stimulatory activity. As a result the antigen not only fails to activate naive T-cells, but causes them to become anergic, or non- responsive.
  • Isolated DCs loaded with tumor antigen ex vivo and administered as a cellular vaccine have been found to induce protective and therapeutic anti-tumor immunity in experimental animals.
  • in pilot clinical trials of DC vaccination for patients with non-Hodgkin's lymphona and melanoma induction of anti-tumor immune responses and tumor regressions have been observed. Timmerman et al, Annal,
  • DCs can be used as a cell vaccine, but they can also be used as an immunomodulating factor in combination with DNA vaccine. Following DNA vaccination, DCs efficiently present vaccine-encoded antigens. Casares et al, J.
  • Plasmid DNA has an adjuvant effect that promotes DC maturation and migration to lymphoid tissue. However, only a very low number of DCs are usually transfected with a direct injection of plasmid DNA, and a very low number of DCs migrate to the site of injection. Lane et al,
  • Th1-type response By triggering a Th1-type response, not only can inflammatory T cells be recruited to sites of infection in order to activate macrophages, but also they attract neutrophils to the infected area by secreting chemokines.
  • Co-delivery of the GM-CSF adjuvant and glycoprotein D antigen boosts immune response during plasmid DNA vaccination with naked DNA.
  • cytokines interleukin-12
  • plasmid DNA encoding cytokines with poly( ⁇ -4-aminobutylglycolic acid) complexes. Maheshwari et al., Mol. Ther., 2(2):
  • the tumor suppressor (antigen) p53 and interleukin12 have been administered via gene delivery in a gene delivery system named "LPD" to initiate cytokine response and inhibit tumor growth .
  • LPD gene delivery system
  • Intravenous injection of plasmids encoding the human FLT-3 ligand increase the number of functional and natural killer cells (NK).
  • NK functional and natural killer cells
  • FLT-3 has been used to induce development of dendritic cells and boost gene expression during a retrovirus-mediated gene vaccination therapy.
  • CD4O and FLT-3 ligands induce dendritic cells and boost gene expression during a retrovirus-mediated gene vaccination therapy. Borges et al. , J. Immunol. 163(3) 1289-97 (1999).
  • the present invention relates to compositions comprising polynucleotides, such as plasmid DNA, DNA, RNA, viruses or vectors, and at least one block copolymer that induce an increased level of production and infiltration of DCs in response to the expression of the gene product encoded by the above DNA, in particular plasmid DNA.
  • polynucleotides such as plasmid DNA, DNA, RNA, viruses or vectors
  • block copolymer that induce an increased level of production and infiltration of DCs in response to the expression of the gene product encoded by the above DNA, in particular plasmid DNA.
  • transgene an encoded exogenous antigen
  • the compositions of the present invention can also be used to generate large amount of dendritic cells both in vitro and in vivo.
  • the current methods of generation, stimulation, and maturation of DCs are extremely difficult and tedious, while the present invention significantly simplifies the process.
  • the muscle is not considered to be a site for antigen presentation because it contains few if any dendritic cells, macrophages, and lymphocytes.
  • the skin and mucous membranes are the anatomical sites where most exogenous antigens are normally encountered.
  • the skin-associated lymphoid tissue contain specialized cells that enhance immune responses. Raz et al, PNAS, 91: 9519-9523 (1994).
  • Anionic polymers such as dextran sulfate and salmon DNA can decrease gene expression in the muscle. Rolland A., Loc. cit.
  • Block copolymers have been used to improve gene expression in muscle or to modify the physiology of the muscle for subsequent therapeutic applications. See U.S. Patent Nos. 5,552,309; 5,470,568; 5,605,687; and 5,824,322.
  • block copolymers can be used in a gel-like form (more than 1 % of block copolymers) to formulate virus particles used to perform gene transfer in the vasculature.
  • block copolymers In the same range of block copolymers concentration (1-10%), it is possible with block copolymer to modify the permeability of damaged muscle tissue (radiation and electrical injury, and frost bite).
  • DNA molecules can be incorporated into cells following membrane permeabilization with block copolymers.
  • block copolymers were used at concentrations giving gel-like structures and viscous delivery systems. These systems are unlikely to enable diffusion of the DNA injected into the muscle, however, thus limiting infusion of the DNA into the myofibers. There is thus a need for compositions and methods increasing efficacy of polynucleotides expression upon administration to a patient, in particular, in the muscle and in the skin.
  • antisense polynucleotides to treat genetic diseases, cell mutations (including cancer causing or enhancing mutations) and viral infections has gained widespread attention.
  • This treatment tool is believed to operate, in one aspect, by binding to "sense" strands of mRNA encoding a protein believed to be involved in causing the disease site sought to be treated, thereby stopping or inhibiting the translation of the mRNA into the unwanted protein.
  • genomic DNA is targeted for binding by the antisense polynucleotide (forming a triple helix), for instance, to inhibit transcription. See Helene, Anti-Cancer Drug Design, 6:569 (1991).
  • an antisense molecule can be designed that binds the sense strand by the Watson- Crick base-pairing rules, forming a duplex structure analogous to the DNA double helix.
  • a serious barrier to fully exploiting this technology is the problem of efficiently introducing into cells a sufficient number of antisense molecules to effectively interfere with the translation of the targeted mRNA or the function of DNA.
  • the invention relates to compositions for inducing activation of dendritic cells comprising a polynucleotide and at least one block copolymer of an alkylether.
  • the present invention relates to methods of activation of dendritic cells comprising administering, particularly intramuscular and intradermal administration, of polynucleotides, such as viruses, RNA, DNA, plasmid DNA or derivatives thereof, and at least one polyoxyethylene-polyoxypropylene block copolymer.
  • polynucleotides such as viruses, RNA, DNA, plasmid DNA or derivatives thereof
  • the block copolymer is present in amounts insufficient for gel formation.
  • the invention also relates to methods of use and compositions comprising at least one polynucleotide or derivative thereof and at least one block copolymer wherein the block copolymer is present at a concentration below about 15% wt/vol.
  • the compositions further comprise a polycation.
  • the compositions also comprise mixtures of block copolymers.
  • the invention also relates to compositions wherein the composition forms a molecular solution or colloidal dispersion, including but not limited to, a suspension, emulsion, microemulsion, micelle, polymer complex, or other types of molecular aggregates.
  • These compositions are useful for increasing the level of production and infiltration for DCs in response to the expression of the gene product encoded by the polynucleotide present in the compositions.
  • the compositions are also useful for increasing the immune response and to generate large amounts of dendritic cells in vivo and in vitro.
  • the invention further relates to methods of delivering polynucleotides to a cell comprising administering to a cell the described compositions.
  • the invention is based in part, on a number of unanticipated surprising discoveries.
  • One is that the infiltration and activation of dendritic cells in vitro increased significantly upon previous exposure of the cells to a composition comprising a polynucleotide and at least one block copolymer.
  • Another is that immunization is improved when polynucleotide molecules (e.g. plasmid DNA and viruses) are formulated with a single or a combination of block copolymers.
  • block copolymers also called "poloxamers”
  • fewer polynucleotide molecules are required to get an immune response, the time to raise the response is shortened, and that there is no need for a booster injection.
  • using fewer polynucleotide molecules will decrease the likelihood of getting polynucleotides integrated into the chromosome(s) of the host organism. Further, using fewer polynucleotides will decrease the likelihood of developing anti-polyucleotide (or anti-DNA) antibodies which have been associated with diseases such as, but not limited to, systemic lupus erythematosus.
  • Backbone Used in graft copolymer nomenclature to describe the chain onto which the graft is formed.
  • Block copolymer A combination of two or more chains of constitutionally or configurationally different features.
  • Branched polymer A combination of two or more chains linked to each other, in which the end of at least one chain is bonded at some point along the other chain.
  • Chain A polymer molecule formed by covalent linking of monomeric units.
  • Copolymer A polymer that is derived from more than one species of monomer.
  • Cross-link A structure bonding two or more polymer chains together.
  • Dendrimer A regularly branched polymer in which branches start from one or more centers.
  • Dispersion Particulate matter distributed throughout a continuous medium.
  • Graft copolymer A combination of two or more chains of constitutionally or configurationally different features, one of which serves as a backbone main chain, and at least one of which is bonded at some points along the backbone and constitutes a side chain.
  • Homopolymer Polymer that is derived from one species of monomer.
  • Link A covalent chemical bond between two atoms, including bond between two monomeric units, or between two polymer chains.
  • Polymer blend An intimate combination of two or more polymer chains of constitutionally or configurationally different features, which are not bonded to each other.
  • Polymer fragment A portion of polymer molecule in which the monomeric units have at least one constitutional or configurational feature absent from adjacent portions.
  • Polynucleotide A natural or synthetic nucleic acid sequence. Repeating unit: Monomeric unit linked into a polymer chain. Side chain: The grafted chain in a graft copolymer.
  • Starblock copolymer Three or more chains of different constitutional or configurational features linked together at one end through a central moiety.
  • Star polymer Three or more chains linked together at one end through a central moiety.
  • Surfactant Surface active agent that is adsorbed at interface.
  • Viral vector A construct derived from a virus and used in gene transfer.
  • the present invention is directed to compositions for activation of dendritic cells comprising at least one block copolymer and compositions comprising at least one polynucleotide or derivative therof and at least one polyoxyethylene- polyoxypropylene block copolymer.
  • the present invention is also directed to methods of inducing the activation of dendritic cells and increasing the immune response of an animal by administrating the compositions.
  • Preferred embodiments include compositions comprising polynucleotides and block copolymers with cationic segments as well as compositions comprising polynucleotides and nonionic polyether block copolymers.
  • polynucleotides are formulated with block copolymers of poly(oxyethylene) and poly(oxypropylene).
  • the preferred compositions of this invention further comprise polycations.
  • compositions of the current invention provide an efficient vehicle for introducing polynucleotides into a cell, protecting polynucleotides against degradation in body fluids, transport of polynucleotides across biological membranes and biological barriers (such as the blood-brain barrier, blood-cerebral fluid barrier, and intestinal barrier), modification of biodistribution of polynucleotides in the body and enhancement of gene expression including selective gene expression in various tissues and organs in the body of human or animal.
  • the invention further relates to methods of inserting or delivering polynucleotides into cells utilizing the compositions of the invention, and methods of treatment comprising administering these compositions to humans and animals.
  • the block copolymer conforms to one of the following formulae:
  • the block copolymers are poly(oxyethylene) and poly(oxypropylene) chain segments.
  • the polynucleotide compositions have polycationic polymers having a plurality of cationic repeating units.
  • the polynucleotides can be complexed with the polycation and stabilized in the complex. These compositions demonstrate increased permeability across cell membranes and are well suited for use as vehicles for delivering nucleic acid into cells.
  • the invention relates to polynucleotide compositions having: (a) a polynucleotide or derivative thereof;
  • the copolymer relates to polymers of formulae: B-A-R A-R A-R-A' and R-A-R'
  • V-b Vl-b
  • Vlll-b Vlll-b
  • Vlll-c Vlll-d
  • A, A', and B are as described above
  • R and R' are polymeric segments having a plurality of cationic repeating units, and each cationic repeating unit in a segment is the same or different from another unit in the segment.
  • the polymers of this embodiment can be termed "polynonionic/polycationic" polymers.
  • Preferred polynonionic/polycationic polymers include polycations that are covalently linked to nonionic polymer segments where the nonionic polymer segments are homopolymer or copolymer of at least one of the monomers selected from the group consisting of acrylamide, gycerol, vinylalcohol, vinylpyrrolidone, vinylpyridine, vinylpyridine N-oxide, oxazoline, or a acroylmorpholine, and derivatives thereof.
  • Nonionic segments comprising products of polymerization of vinyl monomers are also preferred.
  • the R and R', blocks can be termed "R-type" polymeric segments or blocks.
  • the polynucleotide compositions of this embodiment provide an efficient vehicle for introducing polynucleotides into a cell.
  • the invention thus further relates to methods of inserting polynucleotide into cells utilizing the compositions of the invention.
  • the invention relates to polynucleotide compositions comprising a polynucleotide derivative comprising a polynucleotide segment and a polyether segment attached to one or both of the polynucleotide 5' and 3' ends, wherein the polyether comprises an A-type polyether segment.
  • the derivative comprises a block copolymer of formulas:
  • polynucleotide complex comprises a polycationic polymer.
  • the polynucleotide component (pN) of formulas (IX) through (XIII) will preferably have from about 5 to about 1 ,000,000 bases, more preferably about 5 to about 100,000 bases, yet more preferably about 10 to about 10,000 bases.
  • polynucleotide compositions provide an efficient vehicle for introducing polynucleotides into a cell. Accordingly, the invention also relates to methods of inserting polynucleotide into cells the compositions of the invention.
  • polynucleotides are covalently linked to block copolymers of poly(oxyethylene) and poly(oxypropylene).
  • polyetherpolycation copolymers having a polymer, a polyether segment, and a polycationic segment having a plurality of cationic repeating units of formula -NH-R 0 , wherein R° is a straight chain aliphatic group of 2 to 6 carbon atoms, which may be substituted, wherein said polyether segments comprise at least one of an A-type of B-type segment.
  • the polycation polymer has a polymer according to the following formulae:
  • R° is a straight chain aliphatic group having from 2 to 6 carbon atoms, which may be substituted.
  • Each -NH-R 0 - repeating unit in an R-type segment can be the same or different from another -NH-R 0 - repeating unit in the segment.
  • the invention provides a polycationic polymer having a plurality of repeating units of formula:
  • R 8 is:
  • R 9 is a straight chain aliphatic group of 1 to 12 carbon atoms, and R 10 , R 11 , and R 12 are independently hydrogen, an alkyl group of 1-4 carbon atoms.
  • R 9 preferably is 2-10 carbon atoms, more preferably, 3-8 carbon atoms.
  • R 14 preferably includes an intercalating group, which is preferably an acrydine or ethydium bromide group.
  • the number of repeating units in the polymer is preferably between about 3 and 50, more preferably between about 5 and 20.
  • This polymer structure can be incorporated into other embodiments of the invention as an R-type segment or polycationic polymer. The ends of this polymer can further be modified with a lipid substituent.
  • the monomers that are used to synthesize polymers of this embodiment are suitable for use as the monomers fed to a DNA synthesizer, as described below.
  • z has a value of from 2 to 8; and R 8 is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; and (b) at least one straight or branched chained polyether segment having from about 5 to about 400 monomeric units which is: ( ) a homopolymer of a first alkyleneoxy monomer -OCpenH 2n - or
  • Polymers of formulas (I), (II), (III), or (IV) can also be mixed with each other or can be mixed either additionally or alternatively with one or more of the polymers of formula (V-a or b), (Vl-a or b), (Vll-a or b), and (Vlll-a or b) and/or with polynucleotide derivatives of formulas (IX-a,b,c, or d), (X-a,b,c,d,e, or f), (XI), (XII) or (XIII) to provide an efficient vehicle for delivering polynucleotide to the interior of cells.
  • the degree of polymerization of the hydrophilic (A-type) blocks or the hydrophobic (B-type) blocks of formulas (I) - (XIII) can preferably be between about 5 and about 400. More preferably, the degree of polymerization shall be between about 5 and about 200, still more preferably, between about 5 and about 80.
  • the degree of polymerization of the R-type polycation blocks can preferably be between about 2 and about 300. More preferably, the degree of polymerization shall be between about 5 and about 180, still more preferably, between about 5 and about 60.
  • the degree of polymerization of the polycationic polymer can preferably be between about 10 and about 10,000. More preferably, the degree of polymerization shall be between about 10 and about 1 ,000, still more preferably, between about 10 and about 100.
  • the repeating units that comprise the blocks, for A-type, B-type and R-type blocks will generally have molecular weight between about 30 and about 500, preferably between about 30 and about 100, still more preferably between about 30 and about 60. Generally, in each of the A-type or B-type blocks, at least about
  • ether linkages for the purposes of this application, encompass glycosidic linkages (i.e., sugar linkages). However, in one aspect, simple ether linkages are preferred.
  • compositions of the invention are useful for gene therapy purposes, including gene replacement or excision therapy, and gene addition therapy, vaccination, and any therapeutic situation in which a polypeptide should be expressed or down-regulated in the body or in vitro.
  • compositions for intramuscular administration comprise the block copolymers of poly(oxyethylene) and poly(oxypropylene).
  • the invention relates to compositions comprising at least one poly(oxyethylene) and poly(oxypropylene) block copolymer with oxyethylene content of 50% or less, and at least one poly(oxyethylene) and poly(oxypropylene) block copolymer with oxyethylene content of 50% or more, and a polynucleotide.
  • the preferable ratio by weight of the block copolymer with oxyethylene content of 50% or less to the block copolymer with oxyethylene content of 50% or more is 1 :2, more preferably 1 :5. It is preferred that the compositions of this invention do not form gels. It is preferred that the compositions form molecular solutions or colloidal dispersions.
  • the colloidal dispersions include suspensions, emulsions, microemulsions, micelles, polymer complexes, or other types of molecular aggregates are particularly preferred.
  • concentration of the polymers and block copolymers in the polynucleotide compositions is less that 10%, preferably less that 1%, more preferred less than 0.5%, yet more preferred less than 0.1%.
  • Block copolymers are most simply defined as conjugates of at least two different polymer segments (Tirrel, M., Interactions of Surfactants with Polymers and Proteins, Goddard E.D. and Ananthapadmanabhan, K.P. (eds.), CRC Press,
  • A-B-A type triblock Consequent conjugation of more than two segments by their termini yields an A-B-A type triblock, A-B-A-B- type multiblock, or even multisegment A-B-C- architectures.
  • a main chain in the block copolymer can be defined in which one or several repeating units are linked to different polymer segments, then the copolymer has a graft architecture of, e.g., an A(B) n type.
  • More complex architectures include for example (AB) n or A n B m starblocks which have more than two polymer segments linked to a single center.
  • Formulas XVIII - XXIII of the invention are diblocks and triblocks.
  • conjugation of polycation segments to the ends of polyether of formula XVII yields starblocks (e.g., (ABC) 4 type).
  • starblocks e.g., (ABC) 4 type.
  • the polyspermine of examples 13-15 (below) are branched. Modification of such a polycation with poly(ethylene oxide) yields a mixture of grafted block copolymers and starblocks. In accordance with the present invention, all of these architectures can be useful for polynucleotide delivery.
  • the invention provides a polynucleotide complex between a polynucleotide and polyether block copolymers.
  • the polynucleotide complex will further include a polycationic polymer.
  • the compositions can further include suitable targeting molecules and surfactants.
  • the invention provides a polynucleotide complex between a polynucleotide and a block copolymer comprising a polyether block and a polycation block.
  • the invention provides polynucleotides that have been covalently modified at their 5' or 3' end to attach a polyether polymer segment.
  • Preferred polycation polymers and polycation segments of the copolymers include but are not limited to polyamines (e.g., spermine, polyspermine, polyethyleneimine, polypropyleneimine, polybutylene-imine, polypentyleneimine, polyhexyleneimine and copolymers thereof), copolymers of tertiary amines and secondary amines, partially or completely quatemized amines, polyvinyl pyridine, and the quaternary ammonium salts of these polycation segments.
  • These preferred polycation fragments also include aliphatic, heterocyclic or aromatic ionenes. Rembaum et al, Polymer Letters, 6:159 (1968);
  • the polycationic polymers and the R-type blocks have several positively ionizable groups and a net positive charge at physiologic pH.
  • the polyether/polycation polymers of Formulas (V) - (VIII) can also serve as polycationic polymers.
  • the polycation segments have at least about 3 positive charges at physiologic pH, more preferably, at least about 6, still more preferably, at least about 12.
  • polymers or segments that, at physiologic pH can present positive charges with a distance between the charges of about 2A to about 1 ⁇ A. The distances established by ethyleneimine, aminopropylene, aminobutylene, aminopentylene and aminohexylene repeating units, or by mixtures of at least two of these groups are most preferred.
  • polycationic segments that utilize (NCH 2 CH 2 ), (NCH 2 CH 2 CH 2 ), (NCH 2 CH 2 CH 2 CH 2 ), (NCH 2 CH 2 CH 2 CH 2 ), and (NCH 2 CH 2 CH 2 CH 2 CH 2 ) repeating units, or a mixture thereof.
  • the polycation polymers and polyether/polycation copolymers are mixed with polyoxyethylene- polyoxypropylene block copolymers.
  • Oligoamines and conjugates of oligoamines with polyethers can be used in this invention as polycationic molecules, particularly, in mixtures with polyoxyethylene-polyoxypropylene block copolymers.
  • Examples of oligoamines useful in this invention include but are not limited to spermine, spermidine, and other DNA-condensing agents.
  • Ethyleneimine oligoamines such as diethylenetriamine and pentaethylene- hexamine, propyleneimine oligoamines such as N-(3-aminopropyi)-1 ,3- propanediamine and N,N'-b/s-(3-aminopropyl)-1 ,3-propanediamine, butyleneimine oligoamines, pentyleneimine oligoamines, hexyleneimine oligoamines, heptyleneimine oligoamines and derivatives thereof are particularly useful in this invention.
  • Polycation segments having an -N-R 0 - repeating unit are also preferred.
  • R° is preferably an ethylene, propylene, butylene, pentylene, or hexylene chain which can be modified.
  • in at least one of the repeating units in at least one of the repeating units
  • R° is ethylene, propylene, or butylene.
  • the polycation polymers and polycation segments in the copolymers of the invention can be branched.
  • polyspermine-based copolymers are branched.
  • the cationic segment of these copolymers was synthesized by condensation of 1 ,4-dibromobutane and N-(3-aminopropyl)-1 ,3-propanediamine. This reaction yields highly branched polymer products with primary, secondary, and tertiary amines.
  • branched polycations are products of the condensation reactions between polyamines containing at least 2 nitrogen atoms and alkyl halides containing at least 2 halide atoms (including bromide or chloride).
  • the branched polycations are produced as a result of polycondensation.
  • An example of this reaction is the reaction between N-(3-aminopropyl)-1 ,3- propanediamine and 1 ,4-dibromobutane, producing polyspermine.
  • Branched polycation polymers of this type can be represented by the formula:
  • polyethyleneimine represented by the formula: (NHCH 2 CH 2 ) x ⁇ N(CH 2 CH 2 )CH 2 CH 2 ⁇ y
  • cationic dendrimers for example, polyamidoamines can be also used as polycation segments of block copolymers for gene delivery.
  • cationic dendrimers for example, polyamidoamines can be also used as polycation segments of block copolymers for gene delivery.
  • water-soluble polymers that are nontoxic andf nonimmunogenic.
  • polyether polymers that are homopolymers and copolymers of the ethyleneoxy monomer (-OCH 2 CH 2 -) and propyleneoxy
  • polymer segment of use in the present invention is homopolymer or copolymer of
  • oxazoline or a acroylmorpholine, and derivatives thereof. This includes for
  • polyacrylamides examples include polygycerols, polyvinylalcohols, polyvinylpyrrolidones, and
  • nonionic polymer segments including but not limiting to the following nonionic polymer segments and
  • m a value of from 3 to about 10,000.
  • useful polymers pursuant to formulas (V) - (VIII) include the poly(oxyethylene)-poly-L-lysine) diblock copolymer of the following formula:
  • / is an integer of from about 5 to about 100, and / is an integer from about 4 to about 100.
  • a second example is the poly(oxyethylene)-poly-(L-alanine-L-lysine) diblock
  • / is an integer of from about 5 to about 100, and j is an integer from about
  • a third example is the poly(oxyethylene)-poly(propyleneimine/butyleneimine) diblock copolymer of the following formula: O
  • a fourth example is the poly(oxyethylene)-poly(N-ethyl-4-vinylpyridinium bromide) ("pOE-pEVP-Br") of formula:
  • i is an integer of from about 5 to about 100 and j is an integer of from about 10 to about 500.
  • Still another example is the polymer of formula: H-G / -(NH(CH 2 ) 3 ) 2 -N-NH-CO-O-(CH 2 CH 2 O) / CO-G m -(NH(CH 2 ) 3 ) 2 -NH 2
  • Polyoxypropylene-polyoxyethylene block copolymers can also be designed with hydrophilic blocks comprising a random mix of ethylene oxide and propylene oxide repeating units. To maintain the hydrophilic character of the block, ethylene oxide will predominate. Similarly, the hydrophobic block can be a mixture of ethylene oxide and propylene oxide repeating units.
  • Such block copolymers are available from BASF under the tradename PluradotTM. A number of pluronics are designed to meet the following formula:
  • the average numbers of oxyethylene and oxypropylene units were calculated using the average molecular weighs (MW) provided by the manufacturer.
  • the hydrophilic-lipophilic balance (HLB) of the copolymers were determined by the manufacturer (BASF Co.).
  • the critical micellization concentrations (CMC) were determined by the surface tension method described in Kabanov et al, Macromolecules 28: 2303-2314 (1995).
  • the diamine-linked block copolymer of formula (XXVII) can also be a member of the family of diamine-linked polyoxyethylene-polyoxypropylene polymers of formula:
  • hydrophobic/hydrophilic properties of a given block copolymer depends upon the ratio of the number of oxypropylene groups to the number of oxypropylene groups.
  • this relationship taking into account the molecular masses of the central hydrophobic block and the terminal hydrophilic blocks, can be expressed as follows:
  • n (
  • H and H 2 are the number of oxypropylene units in the first and second block copolymers, respectively;
  • L. is the number of oxyethylene units in the first block copolymer;
  • L 2 is the number of oxyethylene units in the second block copolymer;
  • m is the weight proportion in the first block-copolymer; and
  • m 2 is the weight proportion in the second block copolymer.
  • N will range in value from about 0.2 to about 9.0, more preferably between about 0.25 and about 1.5.
  • N value can be expressed as follows:
  • a value N will be used, which value will be the weighted average of n for each contributing copolymers, with the averaging based on the weight portions of the component copolymers.
  • N can be used to estimate the micelle-forming properties of a mixture of copolymers.
  • the use of the mixtures of block copolymers enhances solubility and prevents aggregation of more hydrophobic block copolymers in the presence of the serum proteins.
  • the mixtures comprise poly(oxyethylene)- poly(oxypropylene) block copolymers with the ethylene oxide content of more than 50% solubilize hydrophobic block copolymers with ethylene oxide content of no more than 50%.
  • the mixtures of block copolymers comprise block copolymers with oxyethylene content of 70% or more and at least one block copolymer with oxyethylene content of 50% or less. More particularly PLURONIC® F127 is preferred.
  • the preferred ratio of the hydrophilic and hydrophobic copolymer is at least 2:1 (w/w), preferably at least 5:1 (w/w), still more preferably at least 8:1 (w/w).
  • copolymers other than polyethylene oxide-polypropylene oxide copolymers are used, similar approaches can be developed to relate the hydrophobic/hydrophilic properties of one member of the class of polymers to the properties of another member of the class.
  • one or more block copolymers of poly(oxyethylene)-poly(oxypropylene) are combined so as to have a value for N of from about 0.1 to about 9, more preferably from about 0.25 to about 1.5.
  • the compositions comprises a polynucleotide or derivative thereof and at least one polyethylene- polypropylene block copolymer wherein the block copolymers form a molecular solution or colloidal dispersion (the colloidal dispersion includes, but is not limited to, a suspension, emulsion, microemulsion, micelles, polymer complexes, or other types of molecular aggregates or species).
  • the size of the molecular species formed by the block copolymers is one major parameter determining usefulness of the compositions of the current invention.
  • large particles are eliminated by the reticuloendothelial system and cannot be easily transported to the disease site (see, for example, Kabanov et al, J. Contr. Release, 22, 141 (1992); Volkheimer.
  • Small polymer species are nontoxic, can enter into small capillaries in the body, transport in the body to a disease site, cross biological barriers (including but not limited to the blood-brain barrier and intestinal epithelium), absorb into cell endocytic vesicles, cross cell membranes and transport to the target site inside the cell.
  • the particles in that size range are believed to be more efficiently transferred across the arterial wall compared to larger size microparticles, see Labhasetwar et al, Adv. Drug Del. Res. 24:63 (1997).
  • the small size is essential for successful targeting of such particles using targeting molecules.
  • 1-6 carbon atoms mono- or di-alkylaminoalkyl wherein each alkyl independently has 1-6 carbon atoms, chloro, or chloroalkyl wherein the alkyl has from 1-6 carbon atoms, fluoro, fluoroalkyl wherein the alkyl has from 1-6 carbon atoms, cyano or cyano alkyl wherein the alkyl has from 1-6 carbon atoms or carboxyl;
  • the A- and B- type blocks are at least about 80% comprised of -OR 5 - repeating units, more preferably at least about 90%, yet more preferably at least about 95%.
  • the invention relates to a polynucleotide complex comprising a block copolymer of one of formulas (I) - (XIII) wherein the A-type and B-type blocks consist essentially of repeating units of formula -O-R 5 wherein R 7 is a C to C alkyl group.
  • the block copolymers utilized in the invention will typically, under certain circumstances, form micelles of from about 10nm to about 100nm in diameter.
  • the micelles have translational and rotational freedom in solution, and solutions containing the micelles have low viscosity similar to water.
  • Micelle formation typically occurs at copolymer concentrations from about 0.001 to 5% (w/v).
  • concentration of polycationic polymers and polynucleic acid will be less than the concentration of copolymers in the polynucleotide compositions, preferably at least about 10-fold less, more preferably at least about 50-fold.
  • some of the block copolymers utilized in the invention will form gels. These gels are viscous systems in which the translational and rotational freedom of the copolymer molecules is significantly constrained by a continuous network of interactions among copolymer molecules.
  • microsegregation of the B block repeating units may or may not occur.
  • polymer concentrations for both block copolymers and polyether/polycation polymers
  • polyether/polycation polymers will preferably be below about 15% (w/v), more preferably below about 10%, still more preferably below about 5%. In the first embodiment of the invention, it is more preferred that gels be avoided.
  • the cations will associate with the phosphate groups of the polynucleotide, neutralizing the charge on the phosphate groups and rendering the polynucleotide component more hydrophobic.
  • the neutralization is preferably supplied by cations on R-type polymeric segments or on polycationic polymers.
  • the phosphate charge can also be neutralized by chemical modification or by association with a hydrophobic cations such as N-[1-(2,3-dioleyloxy)-propyl]- N,N,N,-trimethylammonium chloride].
  • polynucleotide complexes In aqueous solution, the charge-neutralized polynucleotides are believed to participate in the formation of supramolecular, micelle-like particles, termed "polynucleotide complexes."
  • the hydrophobic core or the complex comprises the charge-neutralized polynucleotides and the B-type copolymer blocks.
  • the hydrophilic shell comprises the A-type copolymer blocks.
  • the size of the complex will generally vary from about 10nm to about 100nm in diameter. In some contexts, it is practical to isolate the complex from unincorporated components. This can be done, for instance, by gel filtration chromatography.
  • the ratio of the components of the polynucleotide composition is an important factor in optimizing the effective transmembrane permeability of the polynucleotides in the composition. This ratio can be identified as ratio 0, which is the ratio of positively charged groups to negatively charged groups in the composition at physiological pH. If 0 ⁇ 1 , the complex contains non-neutralized phosphate from the polynucleotide.
  • the portions of the polynucleotides adjacent to the non-neutralized charges are believed to be a part of the shell of a polynucleotide complex.
  • the polycationic polymer or R- type segment will have non-neutralized charges, and the un-neutralized portions will fold so that they form a part of the shell of the complex.
  • 0 will vary from about 0 (where there are no cationic groups) to about 100, preferably 0 will range between about 0.01 and about 50, more preferably, between about 0.1 and about 20. 0 can be varied to increase the efficiency of transmembrane transport and, when the composition comprises polynucleotide complexes, to increase the stability of the complex.
  • Variations in 0 can also affect the biodistribution of the complex after administration to an animal.
  • the optimal 0 will depend on, among other things, (1) the context in which the polynucleotide composition is being used, (2) the specific polymers and oligonucleotides being used, (3) the cells or tissues targeted, and (4) the mode of administration.
  • compositions of polynucleotides, cationic copolymer, and a suitable surfactant.
  • the surfactant should be (I) cationic (including those used in various transfection cocktails), (ii) nonionic (e.g., Pluronic or Tetronic), or (/// ' ) zwitterionic
  • Suitable cationic surfactants include primary amines, secondary amines, tertiary amines (e.g., N,N',N'-polyoxyethylene(10)-N-tallow-1 ,3-diaminopropane), quaternary amine salts (e.g., dodecyltrimethylammonium bromide, hexadecyl- trimethylammonium bromide, mixed alkyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, benzalkonium chloride, benzethonium chloride, benzyldimethyldodecylammonium chloride, benzyldimethylhexa- decylammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecyl ammonium bromide, methylbenzethon
  • acyl group dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1 ,2-dioleoyl-3-(4'-trimethylammonio) butanoyl-syn-glycerol, 1 ,2-dioleoyl-
  • Suitable non-ionic surfactants include ⁇ -Alkylphenyl polyoxyethylene ether, ⁇ - alkyl polyoxyethylene ethers (e.g., TritonsTM), sorbitan esters (e.g., SpansTM), polyglycol ether surfactants (TergitolTM), polyoxyethylenesorbitan (e.g., TweensTM), polysorbates, polyoxyethylated glycol monoethers (e.g., BrijTM, polyoxylethylene 9 lauryl ether, polyoxylethylene 10 ether, polyoxylethylene 10 tridecyl ether), lubrol, copolymers of ethylene oxide and propylene oxide (e.g., PluronicTM, Pluronic RTM, TeronicTM, PluradotTM), alkyl aryl polyether alcohol (TyloxapolTM), perfluoroalkyl polyoxylated amides, N,N-bis[3-D-gluconamidopropyl]chol
  • R 1 R 2 R 3 R' are hydrocarbon chains and R., is the longest one), sulfobetaine (R 1 R 2 R 3 N + R'S0 3 " ), phospholipids (e.g., dialkyl phosphatidylcholine), 3-[(3- cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate, 3-[(3-chol- amidopropyl)-dimethylammonio]-1 -propanesulfonate, N-decyl-N,N-dimethyl-3- ammonio-1-propanesulfonate, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane- sulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-1 -propanesulfonate, N- octadecyl-N,N-dimethyl-3-ammonio
  • polynucleotides or nucleic acid molecules can be the polynucleotide component of the compositions. These include viruses, natural and synthetic DNA or RNA molecules, analogs thereof, or derivatives thereof, and nucleic acid molecules that have been covalently modified (to incorporate groups including lipophilic groups, photo-induced crosslinking groups, alkylating groups, organometallic groups, intercalating groups, lipophilic groups, biotin, fluorescent, and radioactive groups, and groups that modify the phosphate backbone).
  • nucleic acid molecules are, but notjimited to, antisense nucleic acid molecules, viruses, viral vectors, gene-encoding DNA
  • the polynucleotides can be nucleic acid molecules encoding a secreted or non-secreted protein or peptide, vaccines or antigens.
  • the nucleic acid component can be any nucleic acid that can beneficially be transported into a cell with greater efficiency, or stabilized from degradative processes, or improved in its biodistribution after administration to an animal.
  • Targeting molecules It will in some circumstances be desirable to incorporate, by noncovalent association, targeting molecules. See for example,
  • the targeting molecules that can be associated with the composition typically have a targeting group having affinity for a cellular site and a hydrophobic group.
  • the targeting molecule will spontaneously associate with the polynucleotide complex and be "anchored" thereto through the hydrophobic group.
  • These targeting adducts will typically comprise about 10% or less of the copolymers in a composition.
  • the hydrophobic group can be, among other things, a lipid group such as a fatty acyl group. Alternately, it can be a block copolymer or another natural synthetic polymer.
  • the targeting group of the targeting molecule will frequently comprise an antibody, typically with specificity for a certain cell surface antigen. It can also be, for instance, a hormone having a specific interaction with a cell surface receptor, or a drug having a cell surface receptor. For example, glycolipids could serve to target a polysaccharide receptor. It should be noted that the targeting molecule can be attached to any of the polymer blocks identified herein, including R-type polymeric blocks and to the polycationic polymers. For instance, the targeting molecule can be covalently attached to the free-terminal groups of the polyether segment of the block copolymer of the invention.
  • Such targeting molecules can be covalently attached to the -OH end group of the polymers of the formulas XVIII, XIX, XX, and XXI, and the -NH 2 end group of the polymers of formulas XVIII (preferably the ⁇ -amino group of the terminal lysyl residue), XX or XXIII, or the -COOH end group of the polymers of formulas XVIII and XIX.
  • Targeting molecules can be used to facilitate intracellular transport of the polynucleotide composition, for instance transport to the nucleus, by using, for example, fusogenic peptides as targeting molecules described by Soukchareun et al, Bioconjugate Chem., 6:43 (1995), or Arar et al, Bioconjugate
  • the polynucleotide component of the compositions can be any polynucleotide, but are preferably a polynucleotide with at least about 3 bases, more preferably at least about 5 bases. Still more preferred are at least 10 bases. Included among the suitable polynucleotides are viral genomes and viruses (including the lipid or protein viral coat). This includes viral vectors including, but not limited to, retroviruses, adenoviruses, herpes-virus, or Pox-virus. Other suitable viral vectors for use with the present invention will be obvious to those skilled in the art.
  • the terms "poly(nucleic acid)" and “polynucleotide” are used interchangeably herein.
  • An oligonucleotide is a polynucleotide, as are DNA and RNA.
  • a polynucleotide derivative is a polynucleotide having one or more moieties (i) wherein the moieties are cleaved, inactivated or otherwise transformed so that the resulting material can function as a polynucleotide, or (ii) wherein the moiety does not prevent the derivative from functioning as a polynucleotide.
  • the present compositions can be used in a variety of treatments. In a preferred embodiment, the compositions are used to induce activation or proliferation of dendritic cells and to increase the immune response in animals by administering the above described compositions.
  • the compositions for inducing activation of dendritic cells comprise a polynucleotide and at least one polyoxyethylene-polyoxypropylene block copolymer.
  • the block copolymers are PLURONIC F127 and L61.
  • the block copolymers are a mixture of about 2% w/v F127 and 0.025% L61.
  • the block copolymers are a 10 fold dilution of PLURONIC F127/PLURONIC L61
  • the block copolymers are a 100 fold dilution of PLURONIC F127/PLURONIC L61.
  • the block copolymers are a mixture of PLURONIC F127 and L61 in a ratio of 8:1.
  • compositions can be used in gene therapy including gene replacement or excision therapy, and gene addition therapy (B. Huber, Gene therapy for neoplastic diseases; B.E. Huber and J.S. Lazo Eds., The New York Academy of Sciences, N.Y., N.Y., 1994, pp. 6-11).
  • antisense therapy targets genes in the nucleus and/or cytoplasm of the cell, resulting in their inhibition (Stein and Cheng, Science 261:1004 (1993); De Mesmaeker et al, Ace.
  • Antigen nucleic acid compounds can be used to target duplex DNA in the nucleus. See Helene and Tolume, Biochim, Biophys.,
  • Catalytic polynucleotides target mRNA in the nucleus and/or cytoplasm. Cech, Curr. Opp. Struct. Biol, 2:605 (1992).
  • GTP-cyclhydrdolase I phenylalanine hydrolase, sarcosine dehydrogenase, glucocerobrosidase, glucose-6-phosphste dehydrogenase), dysotrophin, fibronectin, apoliprotein E, cystic fibrosis transmembrane conductance protein, c- src protein, V(D)J recombination activating protein, immunogenes, peptide and protein antigens ("DNA vaccines”) and the like. More than one plasmid or gene can be expressed according to this invention.
  • This can include at least one gene expressing an antigen and at least one gene expressing a molecule that can activate dendritic cells or other antigen presenting cells and thus serve as an adjuvant for enhanced antigen presentation and induced immune response; e.g. a cytokine.
  • adjuvants include but are not limited to interleukins, such as interleukin-12, Flt3 ligand, GM-CSF, CD4O ligand.
  • the antigen can be any product for which an immune response is produced.
  • either antigen or adjuvant protein can be added in combination with the gene therapy.
  • FLT-3 ligand can be injected in the body with the plasmid or retrovirus encoding the antigen.
  • a MIXTURE OF PLURONIC F127/PLURONIC L61 has the capability to induce NF- ⁇ B-driven genes known like cytokines and chemokines that are to provoke infiltration of dendritic cells.
  • SP1017 has a promoter dependence and seems to favor activation of the transcription factor NF- ⁇ B.
  • DNA constructs driven by CMV promoter or NF- ⁇ B-sensitive element cassette are considerably more responsive to the PLURONIC F127/PLURONIC L61 carrier effect compared to the constructs under
  • NF- ⁇ B also known as RelA, NF ⁇ B3 and NF- ⁇ B p65 subunit
  • RelA, NF ⁇ B3 and NF- ⁇ B p65 subunit is a member of the Rel/NF- ⁇ B family of transcription factors which includes p50, cRel, p52 and RelB.
  • NF- ⁇ B p65 subunit was first isolated from
  • the binding sequence of NF- ⁇ B (KB motifs) is composed of about 10 bases having a common sequence which starts with a cluster of G (guanine) and ends with a cluster of C
  • cytosine (consensus sequence 5'-GGGRNNYCCC-3',).
  • IL-1 interleukin-1
  • TNF tumor necrosis factor
  • Such diseases include, rheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis, ⁇ -thalassemia, ⁇ -thalassemia, carbonic anhydrase II deficiency syndrome, triosephosphate isomerase deficiency syndrome, tetrahydrobiopterindeficient hyperphenylalaniemia, classical phenylketonuria, muscular dystrophy such as Duchenne Muscular Dystrophy, hypersarkosinemia, adenomatous intestinal polyposis, adenosine deaminase deficiency, malignant melanoma, glucose-6- phosphste dehydrogenase deficiency syndrome, arteriosclerosis and hypercholesterolemia, Gaucher's disease, cystic fibrosis., osteopetrosis, increased spontaneous tumors, T and B cell immunodeficiency, high cholesterol, arthritis including chronic rheumatoid
  • compositions can also be used to treat neoplastic diseases including, but not limited to, breast cancer (e.g., breast, pancreatic, gastric, prostate, colorectal, lung, ovarian), lymphomas (such as Hodgkin and non-Hodgkin lymphoma), melanoma and malignant melanoma, advanced cancer hemophilia B, renal cell carcinoma, gliblastoma, astrocytoma, gliomas, AML and CML and the like.
  • breast cancer e.g., breast, pancreatic, gastric, prostate, colorectal, lung, ovarian
  • lymphomas such as Hodgkin and non-Hodgkin lymphoma
  • melanoma and malignant melanoma advanced cancer hemophilia B
  • renal cell carcinoma gliblastoma
  • astrocytoma gliomas
  • AML and CML and the like.
  • compositions can be used to treat (/) cardiovascular diseases including but not limited to stroke, cardiomyopathy associated with Duchenne
  • the present invention is also directed to a method of delivering a polynucleotide to a cell comprising administering a composition of the present invention.
  • the method of delivering a polynucleotide to a cell comprises administering a composition comprising a polynucleotide or derivative thereof and at least one polyethylene-polypropylene block copolymer, wherein the block copolymer is present in amounts insufficient for gel formation.
  • the block copolymer is present at a concentration below about 15% wt/vol, more preferably at a concentration below about 10% wt/vol, and most preferably, in concentrations below about 5%.
  • the composition forms a molecular solution or colloidal dispersion, more particularly, the colloidal dispersion is a suspension, emulsion, microemulsion, micelle, polymer complex or other types of molecular aggregates.
  • Target cells for the delivery of a polynucletide composition are, but not limited to, dendritic cells procaryotic or eucaryotic cells, preferably animal cells, more preferably mammalian cells, and most preferably human cells.
  • Cell targets can be ex vivo and/or in vivo, and include T and B lymphocytes, primary CML, tumor infiltrating lymphocytes, tumor cells, leukemic cells (such as HL-60, ML-3, KG-1 and the like), skin fibroblasts, myoblasts, cells of central nervous system including primary neurons, liver cells, carcinoma (such as Bladder carcinoma T24, human colorectal carcinoma Caco-2), melanoma, CD34+ lymphocytes, NK cells, macrophages, hemotopoetic cells, neuroblastona (such as LAN-5 and the like), gliomas, lymphomas (such as Burkitt lymphomas ST486), JD38), T-cell hybridomas, muscle cells such as primary smooth muscle, and the
  • the polynucleotide compositions of the present invention can be used for treatment of animals, including, but not limited to animals such as chickens, pigs, cows, cats, dogs, horses, fish, shrimp, and preferably to mammals, and most preferably humans.
  • the polynucleotide compositions of the invention can be administered orally, topically, rectally, vaginally, by pulmonary route by use of an aerosol, or parenterally, i.e. intramuscularly, intradermally, subcutaneously, intraperitoneally or intravenously.
  • the preferred routes of administration include, but are not limited to intravenous, oral, intradermal, intramuscularly, subcutaneously ot intraperitonenely.
  • the route of administration is direct injection into the tumor.
  • the polynucleotide compositions can be administered alone, or it can be combined with a pharmaceutically- acceptable carrier or excipient according to standard pharmaceutical practice.
  • the polynucleotide compositions can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the polynucleotide compositions can be combined with emulsifying and suspending agents. If desired, sweetening and/or flavoring agents can be added.
  • sterile solutions of the conjugate are usually prepared, and the pH of the solutions are suitably adjusted and buffered.
  • ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers.
  • Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.
  • diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol.
  • the formulation of the polynucleotides will be without any polycationic moiety since naked polynucleotides itself can be transferred and expressed in muscle without any polycation- containing delivery systems.
  • the muscle has the following features: unique cytoarchitecture, multiple nuclei per myotubes, specific-polynucleotides binding proteins (triadin), and unique nucleocytoplasmic transport.
  • triadin specific-polynucleotides binding proteins
  • Cationic complexes of polynucleotides have been shown to enhance uptake and gene expression in virtually all tissue types but surprisingly the same complexes do not contribute to a better uptake and gene expression in muscle.
  • Plasmid p ⁇ -Gal comprises plasmid pUC19 (available from the Institute of Gene Biology, Russian Academy of Sciences) into which a hybrid of a eukaryotic transcription unit and a £. coli ⁇ -galactosidase has been incorporated. With this plasmid, the efficiency of cell uptake can be measured by measuring ⁇ -galactosidase activity extractable from the treated cells.
  • the copolymer utilized was a triblock copolymer of formula (XIV) wherein x plus z was
  • Pluronic A poly(N-ethyl- 4-vinylpyridinium bromide)
  • pEVP-Br poly(N-ethyl- 4-vinylpyridinium bromide)
  • the NIH 3T3 cells were grown in monolayer culture at 37°C under 5% CO 2 , using a DMEM medium containing 2 mM glutamine and 10% fetal calf serum ("FCS"). Cells were grown in monolayer culture were scraped and prepared for the transaction process by washing three times with fresh medium. Aliquots of washed cells that were to be transformed by the method of the invention were suspended at a concentration of 10 6 cells/ml in Pluronic A transfecting medium. The suspended cells were incubated for 2 hours at 37°C and under 5% CO 2 . The cells were then washed with fresh medium and re-plated.
  • FCS fetal calf serum
  • the repeated cells were incubated for 48 hours in DMEM medium containing 10% FCS. During the incubation, the medium was replaced with fresh medium at 16 hours. After the 48 hour incubation, the cells for each incubation were collected by scrapping, washed with PBS, and resuspended in 100 ⁇ l of 0.2 M Tris-HCL (pH 7.4). The cells were lysed with several freeze/thaw cycles, and centrifuged at an excess of 6,000 x/g.
  • the polycation component of the polynucleotide comprised a copolymer of N-ethyl-4-vinylpyridinium bromide and N-cetyl-4-vinylpyridinium bromide, the monomers incorporated in a molar ratio of 97:3, respectively
  • the block copolymer comprised a triblock copolymer of formula (XIV) wherein x+z was 18, and y was 23 (hereinafter "pEVP-co-pCVP-Br").
  • Pluronic B A Pluronic B transfecting solution of 1 ⁇ g/ml p ⁇ -Gal, 3 ⁇ g/ml PEVP- co-pCVP-Br, and 1% (w/v) Pluronic B was prepared in Example 1. The ratio of polycation basic groups to nucleotide Phosphates was about 7. The weight ratio of Pluronic B to p ⁇ -Gal was about 5 x 10 3 .
  • MDCK cells were plated at 8-10 5 cells per plate onto 90 mm plates and incubated overnight under serum-containing growth medium. The serum containing medium was then replaced with serum-free medium, and the cells were incubated at 37°C, under 5% CO 2 for 24 hours. For the cells to be treated with polynucleotide complex, the medium was then replaced with 5 ml Pluronic B transfecting solution. The cells were incubated, with gentle rocking, at 37°C, under 5% C0 2 In control experiments, cells were transfected with polynucleotide complex, the medium was then replaced with 5 ml Pluronic B transfecting solution.
  • the cells were incubated, with gentle rocking, at 37°C, under 5% C0 2 , for 2 hours.
  • cells were transfected using the calcium phosphate procedure as described above (except that plated cells, not suspended cells, were transfected).
  • the polynucleotic component of the polynucleotic complex was p ⁇ -Gal.
  • the polycation component comprised pEVPBr.
  • the block copolymer comprised an octablock copolymer formula (XVII), wherein i was equal to 10 and j was equal to 12 (hereinafter "Pluronic C" available from BASF).
  • Pluronic C A Pluronic C transfecting solution of 1 ⁇ g/ml p ⁇ -Gal, 4 ⁇ g/ml pEVP-Br, and 1 % (w/v)
  • Pluronic C was prepared as in Example 1.
  • the ratio of basic groups to nucleotide phosphates was 10.
  • the weight ratio of Pluronic C to p ⁇ -Gal was 10 3 .
  • the transfection protocol was the same as that used in Example 2. The results were as follows:
  • the polynucleotide component of the polynucleotide complex was plasmid pBC16, a plasmid encoding tetracycline resistance.
  • a block copolymer according to formula (VI) was used.
  • the block copolymer was a poly(oxyethylene)-poly((N-ethyl-4-vinylpyridinium bromide) of formula (XXI), wherein i was 44, and j was 20.
  • a stock solution of second embodiment polynucleotide complex was prepared consistent with the transfection solutions described above. The ratio of copolymer basic groups to DNA phosphates in the solution was 0.2.
  • a complex of plasmid pTZ19 and a diblock copolymer of formula (XXI) (poly(oxyethylene)-poly((N-ethyl-4vinylpyridinium bromide), wherein i was 44 and j was 20) was formed.
  • the solution of polynucleotide complex dissolved in PBS contained about 4 ⁇ g/ml of plasmid and 2O ⁇ g/ml of diblock copolymer. These amounts resulted in a ratio of base groups in the polycation block to DNA phosphate groups of 5. For control incubations, an equivalent amount of free plasmid was dissolved in buffer.
  • PVUII nuclease was added to solution samples containing free DNA or polynucleotide complex, and the amount of undigested, circular plasmid DNA, after various digestion times, was determined by electrophoresis in a polyacrylamide gel. See Kabanov et al, Biopolymers, 31 :1437-1443 (1991). The results were as follows:
  • a complex containing an oligonucleotide complementary to the transcription initiation site of the HIV-1 tat gene (“anti-tat", comprising GGCTCCATTTCTTGCTC) was prepared using the diblock copolymer of formula (XIX) (polyoxyethylene-poly(L-alanine-L-lysine), wherein i is 44 and j is 8).
  • the oligonucleotide complex was prepared in PBS Buffer (pH 7.0) at a concentration of 0.75 OD 260 / ⁇ l oligonucleotide.
  • the ratio of polycation imino and amino groups to polynucleotide phosphate groups was about 50.
  • the mixture was incubated for one hour at room temperature to allow for the formation of the complex. Then, the complex was purified by gel filtration chromatography on Sephadex G-25 using 0.05 M NaCl as the eluent. The resulting solution of complex exhibited a concentration of 0.11 OD 260 / ⁇ l of oligonucleotide.
  • a comparable solution of uncomplex oligonucleotide was prepared. An aliquot of murine blood plasma (10 ⁇ l) was mixed with an equal volume of oligonucleotide complex solution or a solution of free oligonucleotide. Samples were incubated at 37°C for various time periods.
  • the samples were diluted with water and extracted with a water-saturated mixture of phenol:chloroform (1 :1 ).
  • the aqueous phase of the extraction was isolated, and the oligonucleotide therein was precipitated with 3% lithium Perchlorate.
  • the precipitate was washed with acetone, and then dissolved in 100 ⁇ l of water.
  • Oligonucleotide Stabilization This example examined the stability of the oligonucleotide described in
  • Example 6 when complexed with a diblock copolymer of formula (XX) (polyoxyethylene-poly-propyleneimine/butyleneimine, wherein i is 44 and j is 4-8) was examined.
  • XX diblock copolymer of formula (XX) (polyoxyethylene-poly-propyleneimine/butyleneimine, wherein i is 44 and j is 4-8) was examined.
  • the same methodologies that were applied in Example 6 were applied for this example, except that the oligonucleotide concentration was about 0.13 OD 260 / ⁇ l.
  • the results were as follows:
  • Antisense Cell Incorporation Efficiencies This experiment examined the effectiveness of "anti-MDR", an antisense molecule comprising a 17-chain oligonucleotide of sequence CCTTCAAGATCCATCCC complementary to positions 422-438 of the mRNA encoding the MDR1 gene product, in reversing multi-drug resistance in SKVLB cells.
  • SKVLB cells are multi-drug resistant cells derived from a ovarian cancer cell line.
  • the MDR1 gene has been identified as responsible for the multi-drug resistance in SKVLB cells. Endicott and Ling, Ann. Rev. Biochem., 58:137 (1989).
  • the efficiency of the anti-MDR oligonucleotide in the polynucleotide complex of the invention and when in the free state was compared.
  • the free and completed form of the anti-tat oligonucleotide described above were also used.
  • the polynucleotide complexes were formed with the diblock copolymer of formula (XX) (polyoxyethylenepolypropyleneimine/butyleneimine, where i was 44 and j was 9-10).
  • the complexes were prepared by the procedures described in Example 6.
  • the oligonucleotide concentration in the complex or in the free state was 0.17 OD 260 / ⁇ l.
  • the copolymer was present in the concentration sufficient to define a ratio of polycation block imino and amino groups to oligonucleotide phosphate groups of 10.
  • the SKVLB cells were incubated for 3 days at 37°C under 5% CO 2 in the presence of free or completed oligonucleotide (at a concentration of 20 ⁇ M based on oligonucleotide content). Fresh media including free or completed oligonucleotide was added every 12 hours.
  • the daunomycin cytotoxicity (IC 50 ) with respect to the cells treated as described above was measured using the method of Alley et. al., Cancer Res., 48:589-601. The results were as follows:
  • HSV-1 Herpes Simplex Virus 1
  • AGCAAAAGCAGG an equivalently modified sequence complementary to the RNA produced by influenza virus
  • the oligonucleotides were applied to HSV-1 infected cells in either the complexed or the free state.
  • the complex was formed with the diblock copolymer of formula (XIX) (polyoxyethylene-poly(L-alanine-L-lysine), wherein i was equal to 44 and j was equal to 8).
  • Oligonucleotide complexes were formed as described in Example 6.
  • HSV-1 virus strain L2
  • HSV-1 virus strain L2
  • RPMI-L 640 media containing 10% of fetal calf serum and free or complex oligonucleotide was added to the cell.
  • the cells were then incubated at 37°C under 5% C0 2 for 24 hours.
  • HSV-1 infectivity of the of the cell media was then determined using the patch production method described by Virology, A Practical Approach, Mahy, Ed., IRL Press, Washington, D.C., 1985.
  • the results, utilizing varying concentrations of oligonucleotide, were as follows:
  • this example utilized the same procedures as were utilized in Example 9.
  • the cells utilized were BHK cells, a Chinese hamster kidney cell line.
  • the complexed form of the oligonucleotides was used, the complex was formed with the diblock copolymer of formula (XVII) (polyoxyethylene-poly-L-lysine, wherein i was 44 and j was 30), using the procedure described in Example 6.
  • the concentration of the stock solution of complex was 0.09 OD 260 / ⁇ l.
  • the ratio of polycation block imino and amino groups to oligonucleotide phosphates was 10.
  • the oligonucleotides, in complexed or free form were applied to the cells at a concentration of 3.0 ⁇ M. The results were as follows:
  • Polynucleotide complexes between the block copolymer of formula (XVII) (polyoxyethylene-poly-L-lysine, wherein i was 44 and j was 30) and the Anti-HSV and Anti-Influenza oligonucleotides were formed using the methods outlined in Example 9.
  • the concentration of the stock solutions of complexes was 0.9 OD 260 / ⁇ l.
  • the ratio of polycation block imino and amino groups to oligonucleotide phosphates was 10.
  • Inbred white mice (body weight 6-7g) were infected with HSV-1 (strain CI from Belorussian Res. Inst. of Epidemiol. & Microbiol, Minsk) by intraperitoneal injection of 30 ⁇ l of a virus suspension (titre: 10 "7 LD 50 /ml).
  • a 32 P-labelled 17-mer (GGCTCCATTTCTTGCTC) complementary to the transcription initiation site of the HIV-1 tat gene was utilized in this example.
  • the oligonucleotide was modified at its 5'-end with cholesterol as described by Boutorin et al, Bioconjugate Chemistry, 2: 350-356 (1990).
  • a polynucleotide conjugate of the oligonucleotide was formed with the block copolymer of formula (XX) polyoxyethylene-poly (propyleneimine/butyleneimine), wherein i was 44 and j was 9 to 10).
  • the concentration of the stock solution (dissolved in PBS) of complex was 0.18 OD 260 / ⁇ l.
  • the ratio of polycation block imino and amino groups to oligonucleotide phosphates was 50.
  • mice Male C57/BI/6 mice (weight: 20-24 g; obtained from the Russian Research Center of Molecular Diagnostics and Therapy, Moscow) received 50 ⁇ l intravenous injections of Anti-HIV conjugate or free Anti-HIV, at 0.18 OD 260 / ⁇ l dissolved in PBS. At defined times after the injections, blood sample were taken from the tail vein and the animals were sacrificed. The amount of radioactive material in blood or tissue sample was determined by liquid scintillation counting (after appropriate solubilizations). The results were as follows:
  • the first intermediate was dissolved in 50 ml of 1 ,4- dioxane and reacted with 2.7g (12.5 mmoles) of 1 ,4-dibromobutane. Again, the reaction proceeded for 16 h at 20°C, and the resulting second intermediate was recovered and dried as above.
  • the second intermediate was neutralized with acetic acid to a pH of 7-8 and purified by gel filtration on Sephadex G-25, using an aqueous eluent. Three major polymine fractions were obtained, having apparent molecular weights of 1060, 700 and 500, respectively.
  • Poly(oxyethyleneglycol) (1.5g, M.W. 1500, from Fluka) was dissolved in 8 ml of 1 ,4-dioxane and reacted with 0.17 g (1 mmole) of N,N'-carbonylimidazole (Aldrich Co.) at 20°C for 3 h. The reaction mixture was divided into two parts.
  • Example 13 was then added to the first reaction mixture. The resulting mixture was reacted for 16 h at 20°C with stirring. A polymer of formula (XXIII) was isolated from the second reaction mixture by gel filtration.
  • Example 16
  • the precipitate was dissolved in triethylammonium acetate buffer and purified by reverse-phase high performance liquid chromatography on a Silasorb C18 column (9X250 mm, Gilson, France) developed with an acetonitrile gradient in a 20 mM TEAA buffer (p H 8.5).
  • Oligo A Conjugate was separately dissolved in RPMI 1640 medium (ICN Biomedicals Inc., Costa Mesa, CA) to a final concentration of 0.2 mM (based on oligonucleotide absorbance). These stock solutions were then filtered through 0.22 ⁇ m filters to remove any possible bacterial or fungal contamination. Monolayers of Vero cells were incubated for 1 hour at 37°C in serum-free RPMI 1640 together with various concentrations of Oligo A or Oligo A Conjugate.
  • the monolayers while still exposed to oligonucleotides, were then infected with 1 plaque forming unit per cultured cell of HSV-1 , strain L2 (from the Museum of Virus Strains of the D.I. Ivanovskii Institute of Virology, Russian Academy of Sciences, Russian Federation). This infection method has been described by Vinogradov et al, BBRC, 203:959 (1994). After 8 hours of exposure to virus and oligonucleotides, the medium on the cells was replaced with fresh medium containing 10% FCS.
  • DMT represents a dimethoxytrityl group
  • H-phosphonate groups of the block copolymer were oxidized as described in Example 19 using tetramethylenediamine (Sigma) instead of hexamethylenadiamine, resulting in the formation of phosphonamide bonds between the diamines and the backbone phosphates.
  • a diblock copolymer comprising 12-mer oligonucleotide, 5'-GGTTCCTCCTGU (Oligo A, complementary to the splicing site of the early mRNA of type 1 Herpes
  • Example 17 The experiment was performed exactly as described in Example 17 except that (1 ) the oligonucleotide-BDP copolymer of Example 21 was used and (2) a single concentration of oligonucleotide-BDP copolymer (conjugate) was used (4,4M).
  • polyspermine was obtained by stepwise polycondensation of N-(3-aminopropyl)-1 ,3-propanediamine and 1 ,4- dibromobutane as described in Example 13 and used without conjugating to poly(ethylene glycol).
  • B The polyimine polycation synthesized in A was modified by dansyl chloride to obtain a fluorescent dansyl-labeled substance, purified by thin layer chromatography and a major component of the mixture (over 75% in most batches) was analyzed by electrospray mass-spectrometry in positive charge mode.
  • M+3, and M+4 (667.6, 668.5, 669.6, and 670.5).
  • M+54 Two distinct groups were observed, with M/2H+ and
  • the concentration of primary amino groups in the polyimine polycation synthesized in A was determined by fluorescamine method as described by
  • Linear polycations of polyimine type are synthesized by condensation of a diaminoalkyl and bis-aldehyde in the presence of sodium cyanoborohydride using a modified reductive amination procedure described by Aziz et al, J. Pharmac Exper. Therapeutics, 274:181 (1995).
  • 0.33g of malonaldehyde bis(dimethyl acetal) was added in 10 ml of 0.5 N HCI and stirred for 1 hour at 20 ° C to obtain free bis-aldehyde.
  • 1.27g of N,N'-bis[3-aminopropyl]-1 ,4-butanediamine was added to this solution and pH was adjusted to 5.0.
  • Desalted solution was concentrated in vacuo and then reacted with poly-L-lysine, Mw. 19000 in methanol-water solution for 24 h at room temperature at a molar ratio of poly(ethylene glycol) to free aminogroups of poly-L-lysine 0.7:1.0.
  • the conjugate obtained was purified by gel-permeation column chromatography on Sephadex-50 (fine) (Pharmacia) in water and then by reverse phase chromatography on semi- preparative column (Vydac C18 5u ,10 mmx25 cm) in acetonitrile concentration gradient. This yields a grafted polylysine copolymer at 35% yield in which 50% of free aminogroups are substituted with poly(ethylene glycol) as determined by fluorescamine method.
  • the side product of the reaction (10-15 % yield) was the bis-4,4'-dimethoxytrityl- derivative of poly(ethylene glycol).
  • the number of the primary aminogroups in the synthesized sample of the copolymer was determined using a modified procedure described by Weigele et al. (J. Amer. Chem. Soc, 1972, 94:5927). To 1.5 ml of a sample in 20 mM sodium borate, pH 9.5 (aminogroups concentration up to 100 ⁇ M) 0.25 ml of fluorescamine solution (0.024%, Sigma) in acetone was added and vortexed for 5 min. The measurements have been made on Shimadzu spectrofluorometer at excitation wavelength 384 nm and at 430 to 510 nm emission wavelength range.
  • Extinction coefficient at emission 475 nm was determined as equal to 1.58x10 6 M " 1 .
  • the specific amount of primary aminogroups was 0.69 mmol/g.
  • Example 28 Following the procedure of Example 28 but substituting 24g of poly(ethylene glycol), by the same amount of Pluronic P85 and using a molar ratio of activated Pluronic P85 to free aminogroups of polyethyleneimine 0.3:1.0 there is obtained in 70% yield a grafted polyethyleneimine copolymer in which 11 % of free aminogroups of polyethyleneimine are substituted with Pluronic P85.
  • Example 35 Following the procedure of Example 28 but substituting 24g of poly(ethylene glycol), by the same amount of Pluronic P123 (BASF Co.) and using a molar ratio of activated Pluronic P123 to free aminogroups of polyethyleneimine 0.3:1.0 there is obtained in 30% yield a grafted polylysine copolymer in which 9% of free aminogroups are substituted with Pluronic P123.
  • Pluronic P123 BASF Co.
  • EPM electrophoretic mobility
  • the size of the particles of the complex synthesized in B were determine.
  • the EPM measurements were performed at 25°C with an electrical field strength of 15-18 V/cm using "ZetaPlus" Zeta Potential Analyzer (Brookhaven Instrument Co.) with 15 mV solid state laser operated at a laser wavelength of 635 nm.
  • the zeta-potential of the particles was calculated from the EPM values using the Smoluchowski equation.
  • Effective hydrodynamic diameter was measured by photon correlation spectroscopy using the same instrument equipped with the Multi Angle Option.
  • the sizing measurements were performed at 25°C at an angle of 90°.
  • the zeta potential of this sample was close to zero, suggesting that particles were electroneutral.
  • the average diameter of the particles was 35 nm.
  • Example 39 Stability Against Nuclease Digestion 100 ⁇ g of the complex formed between the PS-dT20 and polyethyleneimine- poly(ethylene glycol) block copolymer obtained in Example 39 was treated by 1 mg of snake venom phosphodiesterase (Phosphodiesterase I from Crotalus adamanteus, 0.024 units/mg, Sigma) for 2 and 18 hrs at 37°C. Reaction mixtures were analyzed by gel permeation HPLC for digested PS-dT20. The digestion of the PS-dT20 in this complex was less than 5%. In contrast, free PS-dT20 treated with the same concentration of enzyme for the same time interval was digested completely.
  • snake venom phosphodiesterase Phosphodiesterase I from Crotalus adamanteus, 0.024 units/mg, Sigma
  • Caco-2 cells originating from a human colorectal carcinoma (Fogh et al. J. Natl. Cancer Inst., 59:221-226, 1977) were kindly provided by R.T. Borchardt (The
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS heat-inactivated fetal bovine serum
  • streptomycin 10 ⁇ g/ml
  • All tissue culture media were obtained from Gibco Life Technologies, Inc. (Grand Island, NY).
  • the cells were grown on collagen coated polycarbonate filter chamber inserts (Transwell, Costar Brand Tissue Culture Products, Contd.; pore size 0.4 ⁇ m; diameter 24.5 mm).
  • Caco-2 cell monolayers were preincubated for 30 min. at 37° C with assay buffer, containing sodium chloride (122 mM), sodium bicarbonate (25 mM), glucose (10 mM), HEPES (10 mM), potassium chloride (3mM), magnesium sulfate (1.2 mM), calcium chloride (1.4 mM) and potassium phosphate dibasic (0.4 mM).
  • assay buffer containing sodium chloride (122 mM), sodium bicarbonate (25 mM), glucose (10 mM), HEPES (10 mM), potassium chloride (3mM), magnesium sulfate (1.2 mM), calcium chloride (1.4 mM) and potassium phosphate dibasic (0.4 mM).
  • the assay buffer in the receptor container was replaced by the fresh one, while the assay buffer in the donor container was replaced by 50 ⁇ M fluorescein-labeled PS-ODN or its complex in the assay buffer.
  • the R123 solutions in the donor container also contained H 3 -labeled manitol, a paracellular marker (Dawson, J. Membrane Biol, 77:213-233 (1977) obtained from DuPont Corp. (Boston, MA).
  • the transport of fluorescein- labeled PS-ODN (or manitol) across Caco-2 cell monolayers was expressed as a percentage of the total fluorescein-labeled PS-ODN (or manitol) accumulated in the receptor chamber to the initial amounts of fluorescein-labeled PS-ODN (or manitol) in the donor chamber.
  • a minimum of three different membranes was studied for each drug composition at multiple time points for each membrane. The results were as follows:
  • Example 41 In vitro transfection of plasmid DNA with various block copolymers-based formulations These experiments are performed in Cos-7 cells and carried out as follows;
  • Cos-7 cells are used and are seeded at 7 x 10 5 per well in 12-well plate (Costar) and allowed to rest 24 hours before transfection (confluenly at 70%).
  • Three ⁇ g of pGL3-Luc SV40 is formulated with the different polymers at various N/P ratios.
  • the transfection mixture is prepared as follows; to an eppendorf tube containing
  • the cells are lysed on ice for 30 minutes and then centrifuged at 13,000g for 2 minutes.
  • the supematents are kept and analyzed for luciferase activity.
  • the assay is performed as follows: 20 Dl of supematent is added to luminometric tubes containing 100 Dl of luciferase substrate. Light emission is measured with a luminometer (Berthold) for a period of 5 seconds. The data is reported in relative light units per second and normalized for proteins with the BiCinchoninic Acid assay kit (Sigma).
  • CMV-Luc 50 ⁇ g
  • oligonucleotides 100 ⁇ g
  • mice 200ul containing various block copolymers-based formulations and injected i.v. into C57BI/6 (6-8 week-old) female mice. Twenty-four hours following the injection the mice are sacrificed to harvest various organs in which luciferase activity is measured or in which oligonucleotide accumulation is determined.
  • luciferase activity is measured or in which oligonucleotide accumulation is determined.
  • DNA DNA
  • tissue grinder Kontes Glass Co.
  • cell lysis buffer Promega Corporation
  • protease inhibitors protease inhibitors
  • the extraction mixture is kept on ice for 30 minutes and then centrifuged at a maximum speed for 2 minutes.
  • the supernatents are kept and analyzed for luciferase activity.
  • the assay is done as follows: 20 ⁇ l of supernatent is added to luminometric tubes containing 100 ⁇ l of luciferase substrate (Promega
  • oligonucleotides For oligonucleotides, the major organs are extracted twice with phenol :chloroform and precipitated with ethanol before quantification. The results show that with conventional liposomal formulation and PEI that gene expression is concentrated in the lungs which is a factor known to increase risks of pulmonary embolism. However, gene expression is redirected to liver when formulated with PEI conjugated to a hydrophobic block copolymer such as P123. In addition, when P123 is used alone, gene expression in various organs is very low except in muscle tissue.
  • oligonucleotides For oligonucleotides, the accumulation is observed is kidneys when a hydrophobic carrier (PEI conjugated to PEG) is used and is redirected to liver when a hydrophobic carrier (P85-PEI/P85) is used.
  • a hydrophobic carrier PEI conjugated to PEG
  • P85-PEI/P85 a hydrophobic carrier
  • Various and a multitude of mixture of block polymers can be prepared to give a wide range of hydrophobic and hydrophylic balances that can redirect gene expression and oligonucleotides accumulation in various regions of the body.
  • each injected muscle is dissected and rapidly homogenized with a tissue grinder (Kontes Glass Co.) in cell lysis buffer (Promega Corporation) supplemented with protease inhibitors.
  • the extraction mixture is kept on ice for 30 minutes and then centrifuged at a maximum speed for 2 minutes.
  • the supematents are kept and analyzed for luciferase activity.
  • the assay is done as follows: 20 ⁇ l of supernatent is added to luminometric tubes containing 100 ⁇ l of luciferase substrate (Promega Corporation). Light emission is measured with a luminometer (Berthold) for a period of 5 seconds. The data is reported in relative light units per second per tibialis anterior.
  • block copolymers improve gene expression measured after 5 days post-injection.
  • the use of a cationic reagent does not improve but inhibited gene expression.
  • the reason of this improvement may lie in the block copolymer's property of changing the surface tension of muscle cells and thus increasing the uptake of plasmid DNA in myotubes.
  • the kinetic experiments are prepared in conditions like that described in example 43 except that the muscles are harvested at day 1 , 2, 3, 4, and 7. As shown in the table below gene expression starts earlier with block copolymers and remained the same over a period of 7 days.
  • PLURONIC® F127 conjugated to spermine is obtained by following the procedure of example 28 but substituting 24g of poly (ethylene glycol) by the same amount of PLURONIC® F127 (BASF Co.) and substituting polyethyleneimine, M.W. 25,000 by spermine (Sigma-Aldrich, St-Louis) and using molar excess of 10 g of spermine per 10 g of poly (ethylene glycol) activated by 1 ,1'-carbonyldiimidazole. This method produced 15 g of spermine conjugated PLURONIC® F127.
  • Example 47B Intramuscular transfection with block copolymer conjugated to spermine
  • PLURONIC® F127 was conjugated to spermine as described in example 47A and used to transfect plasmid DNA into the tibialis anterior of 6-8 weeks old C57BI/6 mice. Mice were kept by groups of 5 and fed ad libidum. Five ug of CMV-driven plasmid DNA encoding luciferase is formulated with F127 conjugated to spermine and injected into the tibialis anterior muscles. The rest of the protocol is as in Example 43. The data are shown in the table below. The data demonstrate that spermine conjugated to F127 and formulated with DNA increase transgene expression compared to naked DNA.
  • Example 47 Intramuscular transfection using block copolymer mixed with spermine
  • PLURONIC® F127 was mixed to spermine and used to transfect plasmid DNA into the tibialis anterior of 6-8 weeks old C57BI/6 mice. Mice were kept by groups of 5 and fed ad libidum. Five ug of CMV-driven plasmid DNA encoding luciferase is formulated with F127 mixed to spermine and injected into the tibialis anterior muscles. The rest of the protocol is as in Example 43. The data are shown in the table below. The data demmonstrate that spermine mixed with Pluronic block copolymer increases the rate of transfection.
  • Example 49 Block copolymers used for gene-based vaccination
  • Block copolymers could be used to raise any humoral and cellular immune response against various antigens associated with diseases (cancer, viral infection, etc.).
  • the following example focuses but not limited to HIV.
  • a block copolymer formulation containing a plasmid DNA construct consisting in gp120 gene of HIV, driven by a cytomegalovirus (CMV) promoter is prepared.
  • a volume of 50 ⁇ l of a block copolymer formulation is prepared containing 5 ⁇ g of gp120 plasmid DNA and 0.01% of block copolymer is injected into the tibialis anterior muscle.
  • CMV cytomegalovirus
  • the muscle is removed from the injection site, and prepared as a cell lysate according to the procedures of example 41 to detect the presence of gp120 by means of ELISA kits (Abbot Labs, Chicago, IL).
  • ELISA kits Abbot Labs, Chicago, IL
  • the ability of gp120 antibody present in serum of the plasmid DNA vaccinated mice to protect against HIV infection is determined by a HT4-6C plaque reduction assay, as follows: HT4-6C cells (CD4 + HeLa cells) are grown in culture in RPMI media (BRL, Gaithersburg, Md.). The group of cells is then divided into batches. Some of the batches are infected with HIV by adding approximately 10 5 to 10 6 infectious units of HIV to approximately 10 7 HT4-6C cells.
  • a plasmid containing the dystrophin gene under control of the Rous Sarcoma virus promoter is prepared from the Xp21 plasmid containing the complete dystrophin coding region and the SV40 poly. 200 ⁇ g of the plasmid in 100 ⁇ l of Dystrophin abnormalities of Duchenne's/Becher Muscular 0.1 % block copolymers solution is injected into the quadriceps the mutant mouse strain lacking the dystrophin gene product (MDX mouse; Jackson labs). Expression of functional dystrophin is monitored 7 days post injection by immunohistochemistry according to the procedures described by Watkins et al.
  • dystrophin expression is localized underneath the plasma membrane of the muscle fiber, so that a cross section of the quadriceps muscle give a fluorescence pattern encircling the cell.
  • dystrophin expression is quantitated by Western blot analysis using the affinity purified anti-60kd antibody.
  • block copolymers are used to improve gene expression in the skin of C57B1/57 (6-7 week-old) female mice kept by groups of 5 and fed ad libidum.
  • Five ug of plasmid pCMV-Luc was formulated with 50 ul of a solution containing a combination of the block copolymers PLURONIC® F127/L61.
  • Plasmid pCMV-Luc was a gift from Dr. Albert Descoteaux from the University of
  • the block copolymers were in a w:w ratio of 8:1 (F127:L61) at a final concentration of 0.01 %W:V.
  • the formulation was injected at the tail base of at least 5 C57BI/57 mice. Seven days later the skin and tissue surrounding the injection site was collected and extracted to monitor the luciferase activity as in Example 42. Luciferase activity was measured as described in Example 42. The following data were obtained and activity levels were compared to those of control mice that received only naked DNA in saline. The results demonstrate that plasmid DNA formulated with a combination of block_copolymers exhibited 20-fold higher levels of Luciferase gene expression than DNA administered without the block copolymer.
  • Block copolymers are used to improve the humoral immune response to the protein encoded by a DNA molecule injected intradermally into C57BI/57 mice (6-7 week-old) female mice kept by groups of 5 and fed ad libidum.
  • the C57B1/57 mice were injected intradermally with 5 ug of pCMV-Bgal (encloding the ⁇ -galactosidase protein) with or without a combination of block copolymers of PLURONIC® F127/L61.
  • the formulation was prepared as described in Example 51. Blood samples were collected 2 and 4 weeks after injection to monitor the humoral immune response specific to ⁇ -galactosidase.
  • the detection of specific anti- ⁇ -galactosidase antibodies was determined by means of an ELISA.
  • the ELISA was performed by allowing the adsortion of ⁇ -galactosidase in
  • the plasmid pCMV-ORF5 formulated with a combination of block copolymers and injected intradermally improved the immune response to the encoded protein.
  • Balb/C mice (6-7 week-old) and kept in groups of 4_ were injected intradermally with 5 ug of the plasmid pCMV-ORF5 (encoding the GP5 protein) with or without a combination of block copolymers.
  • the formulation was prepared as described in Example 51. Blood samples were collected 3 and 5 weeks after inoculation to monitor the humoral immune response specific to GP5. A booster inoculation was given after the first 3 week blood collection.
  • a Single Block Copolymer Improves the Humoral Immune Response to DNA Compositions Administered Intramuscularly
  • Example 52 The data are expressed as the percentage of mice responding to the antigen and the average titers of the responding mice. The data demonstrate that after 2 weeks, none of the mice injected with 5 ug of pCMV-Bgal alone showed an immune response. However, all mouse injected with pCMV-Bgal formulated with PLURONIC®85 showed an immune response. The anti- ⁇ -galactosidase antibody titers of mice injected with pCMV-Bgal formulated with PLURONIC®85 were alwa s hi her than the mice injected with pCMV-Bgal alone.
  • Example 56 A Combination of Block Copolymers Improved the Humoral Immune Response to
  • mice 50 ug of pCMV-Bgal with or without a combination of block copolymers.
  • the formulation was prepared as described in Example 51. Blood samples were collected after 2 and 4 weeks to monitor the humoral immune response specific to Dgalactosidase. The detection of specific antibodies was determined by means of an ELISA as described in Example 52. The data are expressed as the percentage of mice responding to the antigen and the average titers of responding mice. The data demonstrate that when mice receive the DNA formulated with the block copolymers: (1) an additional injection or booster is not needed; (2) that less DNA is required to immunize the mice; and (3) the time to develop the immune response is shorter.
  • a Combination of Block Copolymers Improves the Humoral Immune Response to a Protective Surface Antigen of the PRRSV Virus in Pigs and Mice
  • Pigs and Balb/C, CD1 mice (at least 5 animals and all female) were injected intradermally with an adenovirus containing the ORF5 gene of the PRRSV virus (encoding he GP5 protein) with or without a combination of block copolymers (PLURONIC®_F127/L61) on days 0 and 21.
  • the formulation was prepared as described in Example 51. Fifty days later the animals were challenged with the PRRSV virus. Blood samples were collected at 7 days post- challenge to monitor the humoral immune response specific to GP5. The results demonstrated that only the animals that received the adenovirus formulated with PLURONIC® F127/L61 developed an immunological memory as demonstrated by Western-blot against GP5.
  • Poly(oxyethylene)-poly(oxypropylene) block copolymers were dissolved in the phosphate-bufferred saline, 10 ⁇ M, pH 7.4 (PBS) or in 2.5% solution of bovine serum albumin (BSA) in PBS at the concentrations shown below, and the mixtures incubated for at least one hour at 22.5°C or 37°C.
  • the effective diameters of the aggregates formed in these systems were then measured by quasielastic light scattering method as described by Kabanov et al., Macromolecules 28:2303-2314 (1995). The results were as follows:
  • LPS Liquid Phase Separation
  • hydrophobic poly(ethyleneoxide)- poly(propyleneoxide) block copolymers with propylene oxide content not less than 50% (w/v) reveal tendency for aggregation in aqueous solutions at physiological temperature
  • (2) aggregation and phase separation of these copolymers is significantly enhanced in the presence of serum proteins.
  • hydrophilic poly(oxyethylene)- poly(oxypropylene) block copolymers with ethylene oxide content more than 50% (w/v) prevent aggregation of hydrophobic hydrophilic Poly(oxyethylene)- poly(oxypropylene) block copolymers with propylene oxide content not less than 50% (w/v) at physiological temperatures; (2) hydrophilic poly(oxyethylone)- poly(oxypropylene) block copolymers with ethylene oxide content more than 50% (w/v) prevent aggregation of hydrophobic hydrophilic poly(oxyethylene)- poly(oxypropylene) block copolymers with propylene oxide content not less than 50% in the presence of serum proteins.
  • PLURONIC®F127 is particularly preferred.
  • mice were injected i.m. with 50 ⁇ g of pCMV- ⁇ gal in 50 ⁇ l of saline or 50 ⁇ g of pCMV/bgal in 50 ⁇ l of the solution containing a mixture of PLURONIC
  • the muscle sections were then used to perform an immunohistochemistry analysis in order to determine the cell type infiltrating leukocytes the muscle following the injection.
  • the infiltrating cells were extracted from the muscles to perform flow cytometry studies.
  • Antibodies against CD3, CD4, CD8 and CD11a molecular markers were chosen to determine if the isolated cells were T-cells, antibody against B220 molecule was chosen to determine if the cells were
  • GM-CSF growth factor known to activate the differentiation of DCs
  • GM-CSF + IL-4 and LPS also known to be a stimulant of DCs
  • PLURONIC F127/PLURONIC L61 was used to test its effect on gene expression (transcription) in muscle (tibialis anterior) of C57B1/6 (6-7 week-old) female mice kept by groups of 4 and fed ad libidum. Five ⁇ g of
  • CMV-, 5V40-, AP-1 , NF-kB-driven plasmid DNAs encoding for luciferase are formulated with and without PLURONIC F127/PLURONIC L61 and injected intramuscularly into the tibialis anterior muscle. Before each injection, the mice are anesthetized with a mixed solution of ketamine and xylazine. Mice are sacrificed 5 days following the injection and each injected muscle is dissected and rapidly homogenized with a polytron in cell lysis buffer (Promega Corporation) supplemented with protease inhibitors. The extracts are kept on ice for 30 minutes and then centrifuged at a maximum speed for 2 minutes.
  • the supernatents are kept and analyzed for luciferase activity.
  • the assay is done as follows: 20 ⁇ l of supernatent is added to luminometric tubes containing 100 ⁇ l of luciferase substrate (Promega Corporation). Light emission is measured with a luminometer (Berthold) for a period of 5 seconds. The data is initially in relative light units per second per tibialis anterior but then reported in percentage of increase over naked DNA. As shown in the table below, PLURONIC F127/PLURONIC L61 has a promoter dependence leading to differential transcription.
  • the cells are then extracted in lysis buffer for 30 minutes and then centrifuged at a maximum speed for 2 minutes.
  • the supernatents are kept and analyzed for luciferase activity.
  • the assay is done as follows: 20 ⁇ l of supernatent are added to luminometric tubes containing 100 ⁇ l of luciferase substrate (Promega

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EP1622573A2 (en) * 2003-05-12 2006-02-08 Becton, Dickinson and Company Molecules enhancing dermal delivery of influenza vaccines
US7588774B2 (en) 2003-05-12 2009-09-15 Becton, Dickinson And Company Molecules enhancing dermal delivery of influenza vaccines
JP2009534464A (ja) * 2006-04-24 2009-09-24 ブルース メディカル アクテボラゲット ポリマー系制癌剤

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JP2006131591A (ja) * 2004-11-09 2006-05-25 National Cardiovascular Center 医薬組成物及び該組成物による遺伝子治療方法

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Cited By (6)

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US6875748B2 (en) 2000-04-21 2005-04-05 Vical Incorporated Compositions and methods for in vivo delivery of polynucleotide-based therapeutics
EP1622573A2 (en) * 2003-05-12 2006-02-08 Becton, Dickinson and Company Molecules enhancing dermal delivery of influenza vaccines
JP2007525463A (ja) * 2003-05-12 2007-09-06 ベクトン・ディキンソン・アンド・カンパニー インフルエンザワクチンの真皮送達を向上する分子
EP1622573A4 (en) * 2003-05-12 2008-09-10 Becton Dickinson Co MOLECULES ENHANCING DERMAL DELIVERY OF INFLUENZA VACCINES
US7588774B2 (en) 2003-05-12 2009-09-15 Becton, Dickinson And Company Molecules enhancing dermal delivery of influenza vaccines
JP2009534464A (ja) * 2006-04-24 2009-09-24 ブルース メディカル アクテボラゲット ポリマー系制癌剤

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