EP0831922A2 - Compositions pharmaceutiques ameliorees utilisees pour la therapie genique - Google Patents

Compositions pharmaceutiques ameliorees utilisees pour la therapie genique

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Publication number
EP0831922A2
EP0831922A2 EP96917590A EP96917590A EP0831922A2 EP 0831922 A2 EP0831922 A2 EP 0831922A2 EP 96917590 A EP96917590 A EP 96917590A EP 96917590 A EP96917590 A EP 96917590A EP 0831922 A2 EP0831922 A2 EP 0831922A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
particle
peptide
functional group
synthetic virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96917590A
Other languages
German (de)
English (en)
Inventor
David Robert Thatcher
Roger Kingdon Craig
Paula Elizabeth Wilks
Vincent Trevor Cunliffe
John Hamilton Welsh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Innovata Ltd
Original Assignee
Therexsys Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9513399.7A external-priority patent/GB9513399D0/en
Priority claimed from GBGB9519304.1A external-priority patent/GB9519304D0/en
Priority claimed from GBGB9525955.2A external-priority patent/GB9525955D0/en
Application filed by Therexsys Ltd filed Critical Therexsys Ltd
Publication of EP0831922A2 publication Critical patent/EP0831922A2/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to pharmaceutical compositions useful in the treatment of diseases by gene therapy, and in particular to compositions which rely on nucleic acid condensation agents
  • Gene therapy relies on efficient delivery of DNA to target cells, and expression of the delivered DNA in the nucleus of such cells Different modes of DNA delivery have been proposed, and these involve both viral and non-viral delivery of gene sequences
  • Peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when coadministered with polyiysine DNA complexes (Plank et al. (1994) J. Biol. Chem.269, 12918-24). Trubetskoy et al. (supra) and Mack et al. ((1994) Am. J. Med. Sci. 307, 138-143) suggest that cocondensation of polyiysine conjugates with cationic lipids can lead to improvement in gene transfer efficiency.
  • WO 95/02698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer.
  • Disulfide bonds have been used to link the peptidic components of a delivery vehicle (Gotten et al. (1992) Meth. Enzymol. 2J7, 618-644); see also, Trubetskoy et al. (supra)
  • the chemical modification of the various components, although group specific, is not regiospecific and leads to enormous molecular heterogeneity of the conjugated product.
  • Disulfide bonds are also known to be unstable in biological fluids and thus limits the potency of such compounds in practice.
  • Trubstskoy observed increased efficiency of a conjugate made up of a heterogeneous polyiysine moiety linked through the N-terminus non-specifically to amino functions on a monoclonal antibody
  • an object of the invention is the development of a reproducible and scalable production process for pharmaceutical compositions which facilitate delivery of exogenous DNA to a target cell with high efficiency.
  • Another object of the invention is to provide an improved nucleic acid delivery vehicle that is capable of evading the reticuloendothelial system and thus may withstand degradation in vivo.
  • Yet another object of the invention is to provide a nucleic acid delivery complex that may be designed to target a selected cell population or to target a broad range of cell types.
  • Yet another object of the invention is to provide a nucleic acid delivery complex that has an exceedingly low level of non-specific cell targeting.
  • Yet another object of the invention is to provide a self-assembling virus-like particle, using defined stoichiometry of components.
  • Yet another object of the invention is to provide pharmaceutical formulations for gene delivery which exhibit increased transfection efficiency.
  • the invention encompasses a synthetic virus like particle (i.e., a transfection complex or gene delivery vehicle) for transfecting nucleic acid into a mammalian cell, the synthetic virus like particle comprising a recombinant nucleic acid, a plurality of nucleic acid condensing peptides, the peptides being non-covalently associated with the recombinant nucleic acid such that the nucleic acid is in condensed form, wherein each nucleic acid condensing peptide is a heteropeptide, and the plurality of nucleic acid condensing peptides has low polydispersion.
  • a synthetic virus like particle i.e., a transfection complex or gene delivery vehicle
  • the plural nucleic acid condensing peptides comprise a first nucleic acid condensing peptide and a second nucleic acid condensing peptide, wherein the first nucleic acid condensing peptide comprises a first functional group covalently bound thereto.
  • the first nucleic acid condensing peptide may further comprise a second functional group which may be directly bound to the peptide or may be covalently bound to the first functional group, where the first funcitonal group is bound to the peptide
  • a second nucleic acid condensing peptide also may comprise a second functional group covalently bound thereto, the second functional group bang different from the first functional group.
  • the first and second nucleic acid condensing peptides may have identical or different amino acid sequences.
  • the functional groups which are bound to peptides useful in the invention include a Iigand that is an antigenic peptide or a Iigand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar.
  • the Iigand thus may target cells in a non-specific manner or in a specific manner that is restricted with respect to cell type.
  • the functional group also may comprise a lipid such as palmitoyl, oleyl, stearoyl or cholesterol.
  • the functional group also may comprise a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP).
  • a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP).
  • the functional group also may comprise a fusogenic peptide such as the HA peptide of influenza virus.
  • the functional group also may comprise an enzyme such as a recombinase or an integrase.
  • the functional group also may comprise an intracellular trafficking protein such as a nuclear localization sequence (NLS).
  • an intracellular trafficking protein such as a nuclear localization sequence (NLS).
  • the first functional group may comprise one of a lipid or a neutral hydrophilic polymer such as PEG and the second functional group a Iigand that targets a cell surface receptor.
  • the second functional group when the first functional group comprises a lipid, the second functional group may comprise a Iigand that targets a cellular receptor
  • the first functional group comprises PEG
  • the second functional group may comprise a Iigand that targets a cellular receptor
  • the Iigand may be, for example, one of a sugar moiety or a Iigand whose cellular receptor is restricted to a cell-type, and thus the target cell population may be unrestricted or restricted as to cell type.
  • the first functional group comprises a lipid
  • the second functional group may comprise PEG.
  • the first nucleic acid condensing peptide may comprise 8-24 positively charged amino acid side groups, more preferably the number of positively charged amino acid side groups is 12-18
  • the ratio of positive/negative charges in a synthetic virus like panicle that is capable of targeting a specific mammalian cell type is within the range 0.5-3 per phosphate residue in the nucleic acid; this ratio more preferably being within the range 0.8 - 1.2.
  • the ratio of positive/negative charges in a synthetic virus like particle that is unrestricted with respect to the type of cell it targets is in within the range of 0.5 - 5 per phosphate residue in the nucleic acid, and more preferably within the range of 1.2 - 2.
  • the invention also encompasses a plurality of nucleic acid condensing peptides which are used to formulate a synthetic virus like particle of the invention, wherein each nucleic acid condensing peptide of the plurality being a heteropeptide, and the plurality of nucleic acid condensing peptides having low polydispersion, the peptides being further characterized in that, when contacted with recombinant nucleic acid, the peptides are able to non-covalently associate with the nucleic acid to form a synthetic virus like particle containing condensed recombinant nucleic acid, and the synthetic virus like particle being characterized in that, when contacted with a mammalian cell, the particle can transfect nucleic acid into the cell.
  • the plurality of nucleic acid condensing peptides may comprise a first nucleic acid condensing peptide and a second nucleic acid condensing peptide, wherein the first nucleic acid condensing peptide comprises a first functional group covalently bound thereto, and the second nucleic acid condensing peptide may comprise a second functional group covalently bound thereto, the second functional group being different from the first functional group.
  • a preferred nucleic acid condensing peptide comprises an amino acid sequence of the generic formula
  • each of X 1 is, independently, lysine, arginine, 2.4-diamino-butyric acid or ornithine; wherein each of Y,- 4 is, independently, glutamic acid, alanine, leucine, methionine, glutamine, tryptophan or histidine; wherein each of Z M is, independently, asparagine, glycine, proline, serine, and aspartic acid; wherein B is any one of alanine, glutamic acid or cysteine
  • peptides useful in the invention may contain one or more internal Serine, Threonine, or Cysteine residues, preferably at a position in the sequence which will be exposed for conjugation to a selected Iigand, and thus not on the positively charged (nucleic acid oriented) face of the ⁇ -helix.
  • This positioning of selected reactive amino add residues within the peptide and oriented such that they do not contact the face of the peptide that contacts nucleic acid permits conjugation of the peptide with other functional peptides by bonds of selected and defined stability.
  • Cysteine allows specific conjugation via the thiol side chain to compounds containing other reactive thiol groups (via disulfides), alkylating functions (to form thioether bonds), or other thiol reactive groups such as maleimide derivatives
  • Bonds of "defined stability" are described hereinbeiow, and include bonds such as acid labile bonds (hydrazone) or linkages that are less stable in the reducing environment of the cytosol (disulfide). Such bonds are useful for carrying functional groups on the synthetic virus like particle
  • Preferred peptides which fall within this generic sequence include NBC7 TRRAWRRAKRRAARRCGVSARRAARRAWRRE-OH, and
  • a nucleic acid condensing peptide according to the invention may contain 1) helix- forming amino acids, 2) a repeating three-dimensional structure that contacts the major groove of the nucleic acid, 3) suitable chromophores for quantitation, and 4) a number of "handles" (i e , reactive sites) for regjo-specific conjugation of ligands which form accessory functional domains
  • Nucleic acid condensing peptides of the invention also may include portions of HI (sequence L 13 or LTJ below) which are identified herein as sequences which possess the ability to condense nucleic acid Therefore, a nucleic acid condensing peptide of the invention can comprise a linear combination of the following three consensus sequences where the total sequence length is >17 residues Sequence I
  • K Lysine
  • P Prohne
  • A Alanine
  • X Serine
  • Y Alanine or Valine
  • Z Alanine
  • B Lysine
  • J Alanine or Valine Sequence II
  • X is Alanine or Valine
  • K is Lysine
  • S is Serine
  • P is Proline
  • A is Alanine Sequence HI
  • X is Lysine or Arginine
  • Y is Alanine or Threonine
  • Z is Proline, Alanine or Serine
  • B is Lysine, Threonine or Valine
  • K is Lysine
  • P is Proline
  • A is Alanine
  • a preferred peptide is NBCl, which has the following structure NH 2 -[SV40 NLS]-[Seq I]-[Seq II]-[Seq III]-[SV40 NLS]-[Seq I]-C-COOH, where -C- is Cysteine;where the SV40 NLS has the sequence Pro-Lys-Lys-Lys-Arg-Lys-
  • nucldc acid condensing peptide of the invention will have an amino acid sequence that falls within the following generic sequence:
  • NH 2 -X-(Y)_-C-COOH where X is either absent or Serine or Threonine; Y is sequence I, II or III as defined above; n is 2-6; and C is Cysteine.
  • NBC2 has the structure: NH 2 -[Seq III]-[SV40 NLSl]-[Seq IJ-C-COOH, where -C- is Cysteine.
  • NBC8 has the structure: NH 2 -[Seq I]-[Seq I]-C-COOH where -C- is Cysteine.
  • NBC 13 has the structure: NH [Seq I]-[Seq I]-[Seq I]-C-COOH where -C- is Cysteine.
  • NBC 10 has the structure: NH : -[Seq I]-[Seq I]-[Seq I]-[Seq I]-C-COOH where -C- is Cysteine; the amino acid sequences of which are as follows: NBC2 H-KAVKPKAAKPKIO > KXK lKVEKKSPKKAKKPAAC(Acm)-OH;
  • the invention also features a synthetic virus like particle for transfecting nucleic acid into a mammalian cell, the synthetic virus like particle comprising a recombinant nucleic acid, a first plurality of first nucl c add condensing peptides, each peptide comprising a covalently linked first functional group, a second plurality of second nucleic acid condensing peptides, wherein each nucldc add condensing peptide is a heteropeptide and each plurality of nucleic acid condensing peptides has low polydispersion, wherein each plurality of nucleic acid condensing peptides is non-covalently associated with the recombinant nucleic acid such that the nucleic acid is in condensed form.
  • the synthetic virus like particle may further comprise a third plurality of third nucleic acid condensing peptides, each third peptide comprising a covalently linked second functional group which is different from the first functional group.
  • the first and second functional groups may present in the synthetic virus like particle in a preselected ratio.
  • the first nucleic add condensing peptide may comprise a first functional group covalently bound thereto and may further comprise a second functional group covalently bound thereto.
  • the second functional group may be covalently bound to the first functional group, or it may be covalently bound to the peptide itself.
  • the first and the second nucleic acid condensing peptides may have the same or different amino acid sequences.
  • the invention also encompasses a method of transfecting a mammalian cell with a recombinant nucleic acid, the method comprising contacting a mammalian cell with a synthetic virus like particle as described herein
  • the invention also encompasses a method of transfecting a mammalian cell with a recombinant nucldc add, the method comprising the steps of a) forming a mixture of a synthetic virus like particle of the invention and an endosomal disruption agent, and b) contacting the mixture of step a) with a mammalian cell under conditions sufficient to allow transfection of the cell with the nucleic acid.
  • the invention also encompasses a method of formulating a synthetic virus like particle for administration to a patient, the method comprising mixing a synthetic virus like particle of the invention with an endosomal disruption agent.
  • the endosomal disruption agent is a fusogenic peptide.
  • the invention also encompasses methods of enhancing transfection of a mammalian cell with a recombinant nucleic acid, the method comprising the steps of a) forming a mixture of a synthetic virus like particle and a neutral hydrophilic polymer, as described herein, and b) contacting the mixture of step a) with a mammalian cell under conditions sufficient to allow transfection of the cell with nucleic acid.
  • the invention also encompasses methods of formulating a synthetic virus like particle for administration to a patient, one method comprising mixing a synthetic virus like particle of the invention with a neutral hydrophilic polymer, as described herein.
  • the invention also encompasses a pharmaceutical formulation for administering a recombinant nucleic acid to a patient, comprising a synthetic virus like panicle of the invention in admixture with a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation may further comprise an endosomal disruption agent, or a neutral hydrophilic polymer.
  • the invention also encompasses a method of introducing a recombinant nucleic acid into a patient, the method comprising administering to the patient a therapeutically effective amount of a synthetic virus like particle of the invention or a pharmaceutical formulation of the invention.
  • the invention also encompasses a method of making a synthetic virus like particle for transfecting mammalian cells with a recombinant nucleic acid, the method comprising mixing a recombinant nucleic acid with a plurality of nucleic acid condensing peptides under conditions suffident to permit formation of a synthetic virus like particle containing condensed nucleic acid, wherein the nucleic acid condensing peptide is a heteropeptide and the plurality of nucleic acid condensing peptides has low polydispersion
  • the mixing step comprises mixing the nucleic acid with a plurality of nucleic acid condensing peptides wherein the plurality comprises a first nucleic acid condensing peptide and a second nucleic acid condensing peptide, each nucleic acid condensing peptide being a heteropeptide and each plurality of nucleic acid condensing peptides having low polydispersion, wherein the first nucleic acid condensing peptide comprises a first functional group covalently bound thereto.
  • the invention also encompasses a method of making a synthetic virus like particle for transfecting mammalian cells with a recombinant nucleic acid, the method comprising mixing a recombinant nucleic acid with a first plurality of first nucleic acid condensing peptides and a second plurality of second nucleic acid condensing peptides under conditions sufficient to permit formation of a synthetic virus like panicle containing condensed nucleic acid, wherein each first and second nucldc add condensing peptide is a heteropeptide and each first and second plurality of nucleic acid condensing peptides has low polydispersion.
  • the method also may include the step of mixing the synthetic virus like panicle with an endosomal disruption agent.
  • the first nucldc add condensing peptide may comprise a first functional group covalently linked thereto, and the second nucleic acid condensing peptide may comprise a second functional group covalently linked thereto.
  • the first and the second nucleic acid condensing peptides may have the same or different amino acid sequences.
  • the first and second functional groups also may be the same or different groups.
  • Each nucleic acid condensing peptide may have additional functional groups bound thereto, and these functional groups may be directly bound to the peptide or indirectly bound to the peptide through another functional group.
  • the mixing step of this method of the invention may comprise the step of selecting a ratio of first and second functional groups such that the proportions of first and second nucleic acid condensing peptides having covalently bound thereto first and second functional groups, respectively, that are mixed with recombinant nucleic acid conespond to this ratio.
  • the invention also encompasses high precision chemistry in that the bonding position on a nucleic add condensing peptide may be selected for covalent linkage to a functional group, and the position on the functional group for bonding to an amino acid on a nucleic acid condensing peptide also may be selected
  • the selection of a bonding position on a nucleic acid condensing peptide may include, for example, selecting the amino acid position on the first nucleic acid condensing peptide for covalent linkage to the first functional group and selecting the amino acid position on the second nucleic acid condensing peptide for covalent linkage to the second functional group.
  • the bonding position may be an amino or carboxy terminal amino acid position of the nucleic acid condensing peptide
  • the invention also encompasses a method of making a synthetic virus like particle for transfecting mammalian cells with a recombinant nucleic acid, the method comprising a) contacting a plurality of first nucleic acid condensing peptides with a recombinant nucleic acid in high salt concentration for a time sufficient to allow noncovalent association of the plurality of nucleic acid condensing peptides with the nucleic acid and condensation of the nucleic acid, b) diluting the salt concentration of step a) to a lower salt concentration and bringing the solution to a concentration of neutral hydrophilic polymer which permits stable particle formation.
  • the method also includes an additional step, after or coincident with step b), of adding a plurality of second nucleic acid condensing peptides, each second peptide comprising a lipid group covalently linked thereto, wherein each first and second nucleic acid condensing peptide is a heteropeptide and each plurality has low polydispersion.
  • step b) the dilution is performed simultaneously with the adding of the plurality of second nucleic add condensing peptides.
  • the dilution is performed prior to the adding of the plurality of second nucleic acid condensing peptides.
  • the invention also encompasses a method of making a synthetic virus like particle for transfecting mammalian cells with a recombinant nucleic acid, the method comprising a) forming a mixture of a plurality of first nucleic acid condensing peptides and a recombinant nucleic acid in high salt concentration, b) incubating the mixture of step a) for time sufficient to allow noncovalent assodation of the plurality of nucleic acid condensing peptides with the nucleic acid and condensation of nucleic acid, and c) contacting the mixture of step b) with a plurality of second nucleic acid condensing peptides comprising a lipid group covalently linked to a second peptide, wherein each first and second nucleic acid condensing peptide is a heteropeptide and each plurality of nucleic acid condensing peptides has low polydispersion.
  • Fig. 1(a) is a schematic representation of a synthetic virus like particle according to the invention (10) containing a plurality of a heteropeptide (20a) and condensed nucleic acid (50),
  • Fig. 1(b) is a schematic representation of a virus-like particle according to the invention
  • (11) containing a plurality of a heteropeptide having a first amino acid sequence (20a), a plurality of a heteropeptide having a second amino acid sequence (20b), a plurality of a heteropeptide (21) having a functional group (30) linked thereto, and condensed nucleic acid (50);
  • Fig. 1(c) is a schematic representation of a virus-like particle according to the invention
  • Fig. 1(d) is a schematic representation of a virus-like particle according to the invention
  • Fig 1(e) is a schematic representation of a virus-like particle according to the invention (14) containing a plurality of a heteropeptide (20), a plurality of a heteropeptide having a first functional group (30) linked thereto and a second functional group (31) linked to the first functional group, and condensed nucleic acid (50), and
  • Fig 1(f) is a schematic representation of a virus-like particle according to the invention (f) containing a plurality of a heteropeptide (20), a plurality of a heteropeptide (21) having a first functional group (30) linked thereto, a plurality of a heteropeptide (22) having a second functional group (31) linked thereto, a plurality of a heteropeptide (25) having a third functional group (32) linked thereto, and condensed nucleic acid (50), the first, second, and third funtional groups being present in the particle in a selected ratio
  • FIG 2(a)-(h) are representative electrospray data on the mass distribution of peptides used in the invention
  • Each Figure shows the deconvoluted electrospray mass spectrum Data collected on a VG Instruments Quattro II Instrument fitted with a Quadropole analyzer Peptides were dispensed and diluted into acetonit ⁇ te methyoxyethanol, trifluoroacetic acid and injected directly into the instrument source.
  • Fig 2(a) refers to NBC 1
  • Fig 3 is a gel retardation assay for nucleic acid condensing peptides Complexes were assembled and electrophoresis carried out as described in Example 4 1
  • Fig 4(a) is a graph showing the effect of excess nucleic acid condensing peptide on untargeted transfection by synthetic virus like particles of the invention
  • Fig 4(b) is a graph showing the effect of excess nucleic acid condensing peptide on transfection efficiency.
  • Fig 4(c) is a chart showing the effect of excess nucleic acid condensing peptide on transfection efficiency.
  • Fig. 5 shows the relative transfection potency of individual NBC peptides alone.
  • the assay was performed as follows. Complexes were assembled as described in Example 4.1 using 0.6M sodium chloride, 25mM sodium phosphate buffer ph7.4; and 2 ⁇ g of peptide per ⁇ g of pRSV Luc plasmid DNA. The assay performed as described in Example 4.2.1 using Jurkat Cells (lxlO 6 cells/point) and 2.5 ⁇ g DNA per point.
  • Fig. 6(a) shows the relative transfection efficiency of individual NBC peptides in combination with a fixed amount of Lip 13 in the complex. Experimental details are as described for Fig. 5 except each complex was assembled in the presence of 0.15 ⁇ g Lip 13 per ⁇ g DNA. NBC peptide concentrations were 2 ⁇ g peptide /l ⁇ g DNA.
  • Fig. 6(b) is the same experiment as described in Fig. 6(a) except the ratio of Lip 13 was increased to 0.6 ⁇ g peptide/ 1 ⁇ g DNA.
  • Fig. 7 is a UV absorbance trace of the elution pattern obtained during the ion-exchange chromatography of anti-CD7-NBC2
  • Fig. 8 is a UV absorbance trace of the elution pattern obtained during the ion-exchange chromatography of anti-CD33-NBC2 linked via an acid labile bond and a disulfide linkage.
  • Fig. 9 is a histogram showing reporter gene expression in human myeloid cells (K562 cells) treated with a plasmid containing the reporter gene condensed with anti-CD33-NBCl .
  • the experiment was carried out by the methods described in Examples 4.1 and 4.2.1.
  • the amount of DNA (in a complex) per assay point ( lxlO 6 cells/point) was varied from 0.15-5 ⁇ g.
  • Fig. 10 is a histogram showing the effect of charge ratio on transfection efficiency. Data points are the average of two replicates
  • Fig. 11 is a histogram showing the effect of antibody conjugate (anti-CD7-NBCl) concentration in the nucleic acid condensing on transfection efficiency. Each data point is the average of three replicates.
  • Fig. 12 shows the relative transfection efficiency of targeted complexes (anti CD7/Jurkat cells) assembled with various proportions of ligand-NBC conjugate in the complex.
  • the point of 100% condensation of RSV Luc DNA by anti-CD7-NBCl conjugate and unconjugated NBCl peptides was determined by separate gel retardation analyses.
  • Complexes were then assembled as described in Example 4.1 with various proportions of anti-CD7-NBCl.
  • Conjugated and unconjugated NBCl were added in relative proportions so as to achieve 100% condensation o the nucleic acid, as described in detail in Example 6.
  • Fig. 13 shows the time course for expression of the luciferase reporter gene after transfection of Jurkat cells with anti-CD7-NBCl complexes assembled as described in Example 4.1 in the presence of 0.6M sodium chloride, 2.5 mM HEPES buffer, pH 7.4.
  • Fig. 14 shows the targeting of ludferase reporter gene DNA to HepG2 cells using insulin receptor-mediated gene transfer.
  • Fig. 15 shows that the level of insulin receptor targeted gene transfer is diminished by transfection in the presence of unconjugated Iigand.
  • the control delivery system in this experiment was a non-targeted gene complex composed of 2 ⁇ g NBC 13 per ⁇ g DNA in combination with 0.6 ⁇ g Lip8 per ⁇ g DNA.
  • Fig. 16 shows the transfection of HepG2 cells using mannosylated NBCl/pRSV Luc DNA complexes. Complexes were assembled as described in Example 4.1 and assayed as described in Example 4.2.2 except cells were incubated for both 24 and 90h after transfection.
  • Fig. 17 is a histogram showing increased transfection efficiency obtained if N-palmityl-NBCl is incorporated in the complex Anti CD7-NBC1 conjugate was co-condensed with increasing proportions of Lip2 as described in Example 4.1 and in the legend to Fig. 12 and used to transfect Jurkat cells as described in Example 4.2.1.
  • Fig. 18 is a histogram showing increased transfection efficiency obtained if N-palmityl-NBCl is incorporated in the complex.
  • Anti CD33-NBC1 conjugate was co-condensed with increasing proportions of Lip2 as described in Example 4.1 and in the legend to Fig. 12 and used to transfect K562 cells as described in Example 4.2.1.
  • Fig. 19 is a graph showing the effect of the presence of lipidated nucleic acid condensing peptide on the transfection efficiency of synthetic virus like particles. Complexes were assembled as described in Example 4.1 using NBC2 and Lip2 and assayed using HepG2 cells as described in Example 4.2.2.
  • Fig. 20 shows the effect of Lip 2, Lip 7, Lip 8, and Lip 13 in combination with NBC peptides NBC2, NBC7, NBC8 and NBC 13 on non-targeted transfection of Jurkat cells using RSV Luc luciferase reporter gene DNA.
  • Fig. 21 shows the effect of Lip 13 and palmityl poly-lysine in combination with poly-lysine or NBC 13 on transfection of Jurkat cells using RSVLuc luciferase reporter gene DNA.
  • Fig. 22 is a graph showing transfection of HepG2 and K562 cells using a synthetic virus like panicle.
  • Fig. 23 is a graph showing plasma stability of synthetic vims like particles containing a lipidated peptide.
  • Fig. 24 is a chart showing the effect of varying the concentrations of PEG and salt in a synthetic virus like particle formulation on transfection efficiency.
  • Fig. 25 is a chart showing the effect of varying the concentrations of DNA and salt in a synthetic virus like particle formulation containing 2% PEG on transfection efficiency.
  • Fig. 26(a) shows the relative transfection efficiency of formulations of RSVLuc reporter gene DNA
  • Duplicate 1 shows the effect of the NBC2/Lip2 complex formulated in 0.8M sodium chloride, 25mM HEPES pH 7.4. 2.5 ⁇ g / assay point were diluted directly into lxlO 6 Jurkat cells during the transfection procedure described in Example 4.2.1.
  • Duplicate 2 shows the transfection efficiency if the complex is first diluted into 0.15M sodium chloride and 25mM HEPES.
  • Duplicate 3 shows the transfection efficiency after dilution into the same buffer except containing 10% PEG 10,000.
  • Duplicate 5 shows the effect observed when the complexes were assembled in the presence of 0.8M sodium chloride and 3.5% PEG 10,000 and then diluted as described for Duplicate 3.
  • Fig. 26(b) shows the effect of pre-assembling NBC2 complexes in 0.6M sodium chloride 25mM sodium phosphate and diluting this solution with phosphate buffered saline, phosphate buffered saline containing 0.6 ⁇ g Lip2/ ⁇ g DNA, into phosphate buffered saline containing 10% PEG 10,000 and into phosphate buffered saline containing 10% PEG 1000 and 0.6 ⁇ g Lip 2/ ⁇ g DNA.
  • Fig. 27 shows the effect of maintaining various formulations at either 4° C or -20° C.
  • the formulations S6 - S19 are summarized in Example 13.
  • Fig. 28 is a histogram showing delivery of the synthetic virus like particle to peripheral blood mononuclear cells.
  • Fig.29 is a histogram showing delivery of the synthetic virus like particle to Jurkat cells and peripheral blood mononuclear cells
  • Fig. 30 is a series of photographs of cross-sections of tumor tissue which has been transfected in vivo by a synthetic virus like particle in which transfection by a lacZ reporter gene is indicated as a blue color.
  • the experiments were performed with NBC2/Lip2 complexe assembled and formulated in a high salt formulation as described in Example 12.1.1.
  • the experiments were performed with NBC13/Lip2 complexes assembled and formulated in an isotonic formulation as described in Example 12.2.2.
  • the invention is based on the discovery of a highly efficient synthetic virus-like particle which, when contacted with a mammalian cell, transfers nucleic acid into the cell.
  • the synthetic virus like particle includes a plurality of nucleic acid condensing peptides and condensed nucleic add. The characteristics of these components of the synthetic virus like particle, and how to make and use synthetic virus like particles of the invention are described in detail below.
  • a nucleic acid useful according to the invention may be any form of nucleic acid, e.g., circular, linear, double-stranded, and DNA or RNA.
  • the nucleic acid may be of any length or sequence, e.g., a 10-50 base oligonucleotide or a longer vector DNA, e.g., l-20kb.
  • Nucleic acid condensing peptides useful in the invention possess the following characteristics.
  • Nucleic acid condensing peptides useful in the invention are characterized in that the peptides are heteropeptides.
  • a heteropeptide refers to a peptide having a selected amino acid sequence.
  • the term “heteropeptide” refers to a peptide having an amino acid composition of at least two different amino acids. This is in contrast to a "homopeptide” in which the amino acid composition consists of identical amino acids.
  • a homopeptide consists of 100% of the same amino acid
  • a heteropeptide consists of a polypeptide claim in which as few as a single amino acid differs from the remainder of the amino acids in a chain, or for example 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90%, etc., of the amino acids in a chain differ from the remaining amino acids in the chain.
  • a heteropeptide is therefore a linear peptide consisting of, in terms of its selected amino acid sequence, a variety of (i.e., at least two, at least three, at least four, etc.,) types of amino acids.
  • amino add refers to any of the twenty natural common amino acids, and also may refer to natural, uncommon amino acids or amino acid derivatives or analogs.
  • An amino acid in D- or L- form may be present in a peptide according to the invention.
  • Nucleic acid condensing peptides of the invention are also characterized in that a plurality of nucldc add condensing peptides, or a preparation of nucleic acid condensing peptides useful in the invention has a low polydispersion index.
  • Nuddc add condensing peptide preparations of the prior art e.g., known as polycations, have a polydispersion index >1.1. This is evident in the description of polyiysine peptide preparations in the prior art as having a "mean Mr" (Wu et al., 1987, J. Biol. Chem. 262;4429; Wu et al., 1988, J. Biol. Chem. 263:14621; and Wu et al., 1988, Biochem. 27:887) or having "an average chain length” (Wagner et al., 1991, Proc. Nat. Aca. Sci. 88:4255; Wagner et al., 1992, Proc Nat. Aca. Sci.
  • Such preparations are disclosed as having an average degree of polymerization of a given number of lysine groups, e.g., "polyiysine 90” has an average degree of polymerization of 90 lysine groups; “polyiysine 190” has an average degree of polymerization of 190 lysine groups; etc (Birnsteil et al., supra.)
  • Preparations of polyiysine are available commercially from Sigma Corporation and are known by one of skill in the art and documented upon purchase to have an actual distribution of sizes within each sample which varies per sample, and may vary, for example, from 30% - 150% of the material being distributed.
  • the actual peptide length within such a sample may be, for example, 80% above or 80% below the stated average length of the peptide.
  • the length of the peptide is thus reported as an average of various sizes which average is determined by low-angle light scattering analysis of individual lots of chemically synthesized peptide.
  • Nucldc add condensing peptide preparations useful according to the invention have a low polydispersion index (PDI), also termed index of polydispersity.
  • PDI polydispersion index
  • a "low" polydispersion index refers to a PDI of ⁇ 1.01.
  • Nucleic acid condensing peptides useful in the invention have a PDI ⁇ 1.01, and preferably have a PDI ⁇ 1.001.
  • Nucleic acid condensing peptide preparations of the invention may have a PDI of 1.0 and thus are termed "monodispersed”.
  • a "high" polydispersion index refers to a PDI of >1.1 , >1.2, >1.3, and generally in the range of 1.1 -2.0.
  • the polydispersion index is used to characterize the molecular weight distribution o polymeric compounds.
  • the PDI for a polymer having a homogeneous distribution of molecula wdghts i.e., where the polymer preparation contains a uniform molecular weight
  • the value approaches 2.0.
  • the polydispersion index of a peptide preparation may be determined according to two methods which are described below: 1) using light scattering and colligative properties of the peptide preparation to determine the weight and number average molecular weights of the peptides in a preparation; or 2) using electro-spray mass spectrometry to determine the molecular weights of peptides in a given preparation. It is critical to the invention that the PDI be calculated according to the most accurate of the two methods, i.e., electro-spray mass spectrometry.
  • the former method of calculating the PDI provides only a rough estimate of the PDI in that, for a given peptide preparation, the ratio of Weight Average Molecular Weight / Number Average Molecular Weight for heterogeneous polymers may be determined, respectively, by light scattering and from colligative properties of the peptide preparation (G. Odian in Principles of Polymerisation, John Wiley and Sons, 1981 ) One such colligative property is viscosity (supra).
  • peptide preparations having a PDI ⁇ 1.01 i.e., those peptide preparations synthesized according to methods described herein, a highly accurate measurement of peptide length in a peptide preparation must be provided such that the accuracy is within 0.01% of the mass of each peptide component of the preparation, and preferably within 0.001%.
  • the PDI for polymer peptides disclosed herein may be calculated from analysis of the peptides by electro-spray mass spectrometry. This method gives the exact mass of each component to within 0.001%.
  • Representative electrospray mass sysectometry data of the peptides used in the invention are provided in Fig. 2(a)-(h).
  • the PDI values of the peptide preparations useful in the present invention are in the range of 1.0 - 1.01. Calculation of the PDI from electospray data is illustrated in Example 3.
  • Peptide preparations which are especially useful in the invention possess a PDI ⁇ 1.01, and even ⁇ 1.001.
  • the PDI values for the nucleic acid condensing peptide preparations reported in the prior art are > 1.1.
  • a nucleic acid condensing peptide suitable according to the invention is a basic peptide, i.e., a peptide with net positive charge, for example a peptide or polypeptide comprising 2-50, preferably 12-40, and more preferably 15-38 D or L amino acid residues.
  • the polypeptide includes at least 30%, more preferably 50%, and could include as much as 80-90% basic amino acids, such as lysine, arginine, hisidine, ornithine or a non-natural amino acid containing a side chain having a secondary or tertiary amine group.
  • a nucleic acid condensing peptide according to the invention is preferably a peptide of, for example, 8-50 residues in length which includes a cysteine and/or threonine and/or serine residue which is available for regio-specific conjugation to a Iigand, as defined herein. It is preferred that the cysteine and threonine residues be located N- and C-terminally, respectively, such that they may act as handles for covalent attachment of ligands.
  • nucleic acid condensing peptides useful in the invention also include peptides wherein a large proportion (e.g, 30%-90%) of the amino acid composition of the peptide is a single basic amino acid species, such as lysine
  • a heteropeptide which includes a sequence of from approximately 8 to approximately 30 lysyl residues is useful according to the invention.
  • a peptide which is a homopolymer of lysyl residues is not useful according to the invention because homopolymeric polyiysine tends to be cytotoxic and to stick nonspecifically to cell surfaces under certain conditions
  • the heteropolymeric nucleic acid condensing peptides contemplated herein are advantageous over homopolymers such as polyiysine.
  • a preferred nucleic acid condensing peptide is a peptide or polypeptide having an ⁇ - helical conformation structure.
  • Such peptides have been designed to interact with DNA by interacting through a conformational structure that is alpha helical.
  • a functional group is covalently linked to the peptide, it follows that the functional group of the conjugate may be positioned around the turn of the alpha helical structure so as to be optimally exposed to the outer surface of the virus like particle.
  • DNA-conjugate complexes i.e., DNA noncovalently associated with a conjugate which includes a peptide of the invention covalently linked to a functional group.
  • the relevant intermolecular interactions are refined by building a rigid scaffold which will specifically dock with DNA and allow positioning of the functional moiety such that steric hindrance will not occur.
  • a rigid scaffold is provided by any peptide which forms a stable ⁇ -helix.
  • a number of proteins that specifically interact with DNA interact through an ⁇ -helix structure (10 residue) which lays across the major groove of the DNA helix. This ⁇ -helix is usually followed in the polypeptide by a reverse turn forming the helix-turn-helix motif which is characteristic of these proteins.
  • Most of the interactions that these sequences make with the DNA are weak hydrogen bonds to the phosphate and bases. This is because the function of these domains is to recognize a particular base sequence and then trigger a conformational change. We hypothesized that if such helices are designed so that the hydrogen bonds were replaced by ionic interactions, the binding would be much stronger and useful to us as a docking/condensation peptide.
  • a peptide having an ⁇ -helical scaffold is designed as follows. Using a molecular model of a 10 residue ⁇ -helix, a helical wheel and a model of B-DNA, it is apparent that strong interactions can be made using a repeating sequence motif of X,X 2 -BB-X,X 2 - BB-X,X 2 , where X, is a basic residue interacting with the phosphate on one side of the major groove and X 2 is a basic residue interacting with a phosphate diametrically opposite the other side of the major groove. Either of X, and X, may be, for example, Lysine, arginine, or Histidine.
  • Lysine, Arginine, and Histidine are preferred because both amino acids 1) are of sufficient size to bridge the major groove gap, 2) will interact strongly with phosphate and 3) are strong helix formers. If the residues BB are also residues with strong helix-forming propensities then the peptide will form a stable ⁇ -helix in solution
  • nucleic acid condensing peptides according to this aspect of the invention will fall within the generic formula
  • X is a naturally occurring or synthetic amino acid carrying a positively charged group on the side chain such as lysine, arginine, 2.4-diamino-butyric acid, histidine, and omithine or a non-natural amino acid containing a side chain having a secondary or tertiary amine group; where Y is naturally occurring amino acid which has a high propensity to promote alpha helix formation as defined by Wilt and Thornton (1988) J. Mol. Biol 203.
  • 2221-232 such as Glutamic acid, Alamne, Leucine, Methionine or Glutamine, Tryptophan or Histidine; where Z are naturally occurring amino acids with at least 3 members of the sequence having a high propensity to form stabilized turn stmctures as defined by Wilt Thornton (loc.
  • peptides useful in the invention may contain one or more internal Serine, Threonine, or Cysteine residues, preferably at a position in the sequence which will be exposed for conjugation to a selected Iigand, and thus not on the positively charged (DNA oriented) face of the ⁇ -helix. This positioning of selected reactive amino add residues within the peptide and oriented such that they do not contact the face of the peptide that contacts DNA permits conjugation of the peptide with other functional peptides by bonds of selected and defined stability.
  • Cysteine allows specific conjugation via the thiol side chain to compounds containing other reactive thiol groups (via disulfides), alkylating functions (to form thioether bonds), or other thiol reactive groups such as maleimide derivatives.
  • Bonds of "defined stability" are described hereinbelow, and include bonds such as acid labile bonds (hydrazone) or linkages that are less stable in the reducing environment of the cytosol (disulfide). Such bonds are useful for carrying functional groups on the synthetic vims like particle.
  • a peptide will contain: 1) helix-forming amino acids, 2) a repeating three-dimensional structure that contacts the major groove of the DNA, 3) suitable chromophores for quanthation, and 4) a number of "handles" (i.e., reactive sites) for regio-specific conjugation of ligands which form accessory functional domains.
  • suitable chromophores for quanthation
  • a number of "handles" i.e., reactive sites
  • ligands which form accessory functional domains.
  • Examples of such peptides include NBC7 and NBCl 1.
  • the threonine at position 1 is available for oxidation to glyoxal and therefore for conjugation via an oxime bond
  • tryptophan at positions 5 and 28 are chromophores which will allow quantitation in the presence and absence of DNA.
  • the lysine at position 9 of NBC7 and the glutamic acid at position 9 of NBC- 11 are available for regio-specific conjugation via reductive amination, peptide bonds, etc., and the cysteine at position 16 for conjugation via thioether and disulfide bonds.
  • the C-terminal glutamic acid of NBC7 is available for modification to a hydrazide and therefore for coupling via an oxime or hydrazone bond.
  • Another preferred nucleic acid condensing peptide is a peptide comprising a sequence which is derived from a sequence of histone HI protein and other human histones.
  • Histone HI is a highly basic protein of the histone family that is found in all higher organisms. Histone HI unlike the majority of other members of this family does not assemble as an integral part of the nucleosome Histone HI is believed to interact primarily with those stretches of chromosomal DNA linking the nucleosome complexes of chromatin (Allen et al. Nature 288(1980). 675). Histone HI binds to naked DNA with the same salt dependence as HI depleted chromatin (Kumar and Walker (1980) Nucleic Acids Research 8, 3135).
  • Histone HI protein is not useful as a component of a gene therapy delivery vehicle because it is not readily available and is a biological repressor of its own transcription. Recombinant histone HI cannot readily be produced by recombinant methods, and the protein is too large to be synthesized chemically. Purification of HI from mammalian sources other than human cells poses a safety hazard. The use of histone HI to promote the condensation of plasmid DNA for transfection is likely to reduce expression of the delivered gene because HI is part of a general repressor mechanism where the presence of excess histone HI leads to reduced transcription (Weintraub, H.[1985] cell, 705-71 1 ; Croston et al. [1991] Science 251, 643).
  • Nucleic acid condensing peptides of the invention may include those portions of HI (sequence I, II or III below) which are identified herein as sequences which possess the ability to condense nucldc add Therefore, a nucleic add condensing peptide of the invention can comprise a linear combination of the following three consensus sequences where the total sequence length is >17 residues: Sequence I:
  • K is Lysine, P is Proline; A is Alanine; X is Serine, Threonine or Proline; Y is Alanine, Proline, or Valine; Z is Alanine, Threonine, Lysine, or Proline; B is Lysine, Alanine, Threonine or Valine; and J is Alanine or Valine Sequence II
  • X is Alanine or Valine
  • K is Lysine
  • S is Serine
  • P is Proline
  • A is Alanine.
  • X is Lysine or Arginine
  • Y is Alanine, Valine, or Threonine
  • Z is Proline, Alanine or Serine
  • B is Alanine, Lysine, Threonine or Valine
  • K is Lysine
  • P is Proline
  • A is Alanine.
  • Sequence IV is a consensus sequence from all human histone sequences: Sequence IV
  • A is preferably Lysine or Threonine; B is preferably Glycine or Glutamine; C is preferably Glycine, but can also be Aspartate, Glutamate, or Serine; D is preferably Glycine, but can also be Lysine, Valine, Glutamine, or Threonine; E is preferably Lysine or Alanine; F is preferably Alanine or Lysine, G is preferably Arginine, but can also be Valine or Isoleucine; H is preferably Alanine, but can also be Threonine, Histidine, or Proline; I is preferably Lysine, Arginine, or Glutamine; J is Alanine or Anginine; and K is preferably Lysine or Glutamine.
  • a preferred consensus sequence is:
  • NBCl has the following structure: NH 2 -[SV40 NLS]-[Seq I]-[Seq II]-[Seq III]-[SV40 NLS]-[Seq IJ-C-COOH where -C- is Cysteine;where the SV40 NLS has the sequence Pro-Lys-Lys-Lys-Arg-Lys-Val-Gln- (Dingwall and Laskey (1991) Trends Biochem. Sci. 16, 478).
  • nucleic acid condensing peptide of the invention will have an amino acid sequence that falls within the following generic sequence:
  • NBC2 has the structure: NH 2 -[Seq III]-[SV40 NLSl]-[Seq Ij-C-COOH, where -C- is Cysteine.
  • NBC8 has the structure: NH,-[Con Seq I]-[Con Seq I]-C-COOH where -C- is Cysteine.
  • NBC13 has the structure: NH 2 -[Seq I]-[Seq I]-[Seq lj-C-COOH where -C- is Cysteine.
  • NBC10 has the structure: NH,-[Seq I]-[Seq I]-[Seq I]-[Seq I]-C-COOH where -C- is Cysteine.
  • Synthetic vims like particles prepared with NBCl or NBC2 peptides are insoluble without salt at >10 ⁇ g/ml; therefore, these peptides are preferred where salt is present during particle formulation.
  • the particles are conformationally pure and the peptide/DNA interactions stable, then this salt effect is not observed (charged particles tend to repel each other).
  • h is preferred according to the invention, as described in detail hereinbelow, that some salt be present during formulation of a synthetic vims like particle of the invention.
  • Sequences NBC8 - 10 are derived from part of NBC2 but lack the nuclear localization sequence and have a repeat motif (seq. I, II or III above) which has enabled us to look at the effect of peptide length on function.
  • NBC8 has a double repeat of this sequence, NBC13 a triple repeat and in NBC 10 the sequence is quadmpled.
  • Nucldc add condensing peptides of the invention may contain one or more covalently linked functional groups
  • a "functional group” refers to a protein, peptide, lipid, or chemical group that is covalently linked to a DNA binding peptide, as defined herein, and which has a biological function with respect to particle stability in biological fluids, entry into a cell, or delivery of DNA to the cell nucleus, or integration into the chromosome.
  • the covalent linkage may be a stable or labile linkage, as defined hereinbelow Where the functional group is a peptide, the covalent linkage may be a peptide bond, thus creating a fusion protein.
  • Examples of functional groups useful according to the invention include but are
  • cognate receptor on the surface of a target cell b) a lipid; c) a neutral hydrophilic polymer; d) an endosomal disruption agent; e) an enzyme, and f) an agent which promotes intracellular trafficking into the nucleus, and combinations thereof.
  • a Iigand will include an antigenic peptide recognized by a cell as foreign (for example, a dendritic cell) and thus will promote uptake of the synthetic vims like particle by the cell.
  • a Iigand also may be a targeting molecule having a cognate receptor on the surface of a target cell.
  • a targeting molecule having a cognate receptor on the surface of a target cell.
  • lectins including but not limited to antibodies; lectins; sugars such as monosacharrides or oligosacharrides, for example, mannose, galactose, fucose, and sialic acid; transferrin; and asialoglycoprotein.
  • antibodies which target cells include but are not limited to anti- integrins for targeting keratinocytes, anti-E-selectin for targeting endothelial cells, anti-CD2, CD4 or CD8 for targeting Tcells, anti CD33 for targeting monocyte/macrophage/dendritic cell precusors, anti-HLA Class II (constant region) for targeting macrophages, Bcells and activated dendritic cells, anti-CD80, CD 19 or CD22 for targeting Bcells; and antibodies for targeting cancer cells include but are not limited to anti PEM (polymorphic epithelial mu in) for colon and breast cancers, anti CEA (carcinoembryonic antigen) colorectal tumors, anti MAGE for melanomas, and anti-EGFR-! (epidermal Growth factor receptor- 1) for lung and breast cancers.
  • anti-integrins for targeting keratinocytes
  • anti-CD2, CD4 or CD8 for targeting Tcells
  • anti CD33 for targeting mon
  • the most useful targeting ligands are monoclonal antibodies or a receptor molecule, such as insulin or epidermal growth factor, or alternatively the binding domain of a receptor binding molecule such as that of E-selectin.
  • One type of targeting Iigand useful according to the invention comprises the protein hormone insulin or a derivative of insulin to direct the synthetic vims like particle to cells expressing the insulin receptor, where the insulin or insulin derivative retains receptor binding properties when conjugated to a nucleic acid condensing peptide.
  • the synthesis of insulin and insulin derivative conjugated peptides is described in Example 1, 6 and 7, and the use of such peptides for targeted gene transfer to insulin receptor-bearing cells is described in Example 6.
  • the effidency of transfer of nucleic acid from the synthetic vims like particle to the target cell may be dependent on the density of the Iigand in the synthetic vims like particle.
  • Receptor-ligand interactions which trigger endocytosis usually involve initial oligomerization of the membrane bound receptor. Therefore, it is prefened that the synthetic vims-like particle described herein be used in an amount effective to allow for clustering of bound receptor at the cell surface.
  • One way in which such clustering is achieved is to synthesize a peptide containing a clustered Iigand, such as a clustered monoclonal antibody, as described in Example 6.
  • the targeting Iigand can also be a sugar residue coupled directly to the amino group of an amino add of a nucldc add condensing peptide or indirectly linked to the peptide, for example, through a PEG functional group. Syntheses and uses of such derivatives are described in Examples 1, 6, and 11.
  • Functional groups of the invention also include lipids and thus, when conjugated to a peptide of the invention, form Iipid-derivatized nucleic acid condensing peptides.
  • lipid refers to a four - thirty carbon molecule that is insoluble in water and soluble in alcohol.
  • the term includes fats, fatty oils, essential oils, waxes, sterols, cholesterols, phospholipins, glycolipins, sulfolipins, aminolipins, chromolipins, and fatty acids.
  • the nucleic acid condensing peptide can be specifically modified according to the invention by condensation with an lipid, for example, an activated ester of a fatty acid.
  • the fatty acid is ideally either palmitic acid, oleic acid, such as dioleoylphosphatidylethanolamine, myristic acid, or cholesterol, although other fatty acids, such as stearic acid, may also be employed.
  • An example of the synthesis of N-palmitoyl-NBC2 (Lip 2) is provided in Example 1 and 7, and its use in gene transfer is described in Example 7.
  • the effect of the presence of the lipid component on transfection effidency of the synthetic vims-like particle is dramatic (see results presented in Fig 17-23) leading to >40-fold increase in activity.
  • the phenomenon is unrelated to the effect observed during DNA transfection using cationic lipids, where activity is proportional to the level of cationic lipid in the complex, in which the Iigand density required for transfection is orders of magnitude higher (e.g., lOx-lOOx) than the Iigand density required for gene transfer according to the present invention.
  • Cholesterol refers to a lipid having the carbon ring structure shown in Example 1 and also includes derivatives having attached groups
  • lipidated peptides in the particle or the particle formulation are resistance of the particle to degradation
  • the presence of lipidated peptide in the particle formulation confers higher resistance to inactivation by human plasma
  • Experiments presented in Example 7 show that the presence of lipidated peptide in the particle formulation increases the relative level of transfection after exposure of the synthetic vims like particle to various levels of human plasma in the transfection medium (Fig. 23).
  • stabilizing additives are brought about by the thermodynamically unfavorable interaction of the protein and solvent additive.
  • This tendency of the protein surface to repel the stabilizing additive from the immediate vicinity of that surface is a phenomenon called selective hydration or negative binding. That is, the concentration of this additive near the protein surface is much lower than in the bulk solvent.
  • the surface area of the polypeptide that is in contact with the solvent increases enormously. Because the preferential exclusion of the solvent additive from the solvent layer in contact with the protein is clearly proportional to surface area, the native compact state will be favored over the denatured (extended) state as less solvent additive will have to be preferentially excluded from the native state.
  • Compounds which have this effect are those which typically do not have strong polar or ionic interactions with the protein, do not have strong hydrogen bonding functionalities and have a tendency to repel non-polar molecules
  • Such compounds are polyols, such as sugars, glycerol and hydrophilic polymers such as polyetheylene glycol, methyl cellulose, poly vinyl alcohol, polyvinylpynolidine, hydroxy propyl cellulose, pullulans, polyoxamers, polyoxamines, polysorbates, and poly (2-hydroxy propyl) methacrylamide.
  • hydrophilic polymers polyethylene glycol [PEG], pluronic polyols, polyvinyl alcohol, polyvinylpynolidine
  • PEG polyethylene glycol
  • neutral hydrophilic polymer includes those polmeric molecules which act as co-solvents with peptides of the invention, including polyols, such as sugars, glycerol and hydrophilic polymers such as polyetheylene glycol, methyl cellulose, poly vinyl alcohol and poly- vinylpynolidine, hydroxy propyl cellulose, pullulans, polyoxamers, polyoxamines, polysorbates, and poly (2-hydroxyl propyl) methacrylamide.
  • polyols such as sugars, glycerol and hydrophilic polymers
  • polyetheylene glycol such as polyetheylene glycol, methyl cellulose, poly vinyl alcohol and poly- vinylpynolidine, hydroxy propyl cellulose, pullulans, polyoxamers, polyoxamines, polysorbates, and poly (2-hydroxyl propyl) methacrylamide.
  • polyols such as sugars, glycerol and hydrophilic polymers
  • polyetheylene glycol
  • neutral hydrophilic polymer is used herein as in two forms. That is, a neutral hydrophilic polymer may be used according to the invention as a functional group which is, directly or indirectly, covalently linked to a peptide useful in the invention, or it may be used as a co-solvent during formulation of a synthetic vims like particle (see below). Without being bound to any one theory, it is believed that either use of a neutral hydrophilic polymer according to the invention results in better nucleic acid compaction, smaller overall size, and greater stability of the synthetic virus like particle, and thus produces better transfection efficiency of the particle with respect to target cells.
  • a neutral hydrophilic polymer may be linked to a peptide, and thus presented as a functional group, as described in detail in the Examples d) A functional group which is an endosomal disruption agent.
  • a functional group useful according to the invention includes a Iigand which serves to promote uptake of the synthetic vims like particle by a cell.
  • Iigand which serves to promote uptake of the synthetic vims like particle by a cell.
  • Many peptide molecules are known in the art which have the ability to promote uptake of a molecule into the cell, e.g., by dismpting membrane stmcture.
  • the most useful of such peptides for synthetic vims like particles are those which undergo a pH dependent conformational change, such as the HA peptide from the influenza vims. The stmcture and synthesis of such a peptide is provided in Example 8.
  • fusogenic peptide from Sendai Vims has the amino acid sequence:
  • the fusogenic peptide sequence from HIV gp41 protein H-Ala-Val-Gly-De- ⁇ y-Ala-Leu-Phe-Leu-Gly-Phe-Leu-Gly-Ala-Ala-Gly-Ser-Thr-Met-Gly-Ala- Arg-Ser-OH (M. Rafalaski, J.D. Lear and W.F. DeGrado, Biochemistry 1990,29,7917-7922).
  • the fusogenic peptide sequence from Paradaxin H-Ala-Val-Gly-De- ⁇ y-Ala-Leu-Phe-Leu-Gly-Phe-Leu-Gly-Ala-Ala-Gly-Ser-Thr-Met-Gly-Ala- Arg-Ser-OH (M. Rafalaski, J.D. Lear and W.F. DeGrado, Biochemistry 1990,29,7917-7922).
  • a mechanism by which a fusogenic peptide is believed to promote uptake of a synthetic vims like particle by the cell is as follows. At neutral pH the peptides have a random stmcture and little or no interaction with the cell membrane. The peptides enter the endosomal compartment by diffusion or preferably by being carried in as part of the vims like particle. As the pH of the endosome drops, the HA peptide forms an ⁇ -helical stmcture or aggregates thereof and these insert into the endosomal membrane and dismpt its integrity.
  • a functional group according to the invention may be an enzyme.
  • an enzyme is defined as a molecule which, when covalently linked to a nucleic acid condensing peptide of the invention, is capable of effecting a biological activity involving breakage and reformation of a covalent bond.
  • functional groups include a recombinase or an integrase, both of which promote recombination of the recombinant DNA with cellular DNA, or an intracellular trafficking enzyme, or reporter enzyme such as horseradish peroxidase, as described in Example 9.
  • a functional group according to the invention may be a nuclear localization sequence.
  • Nuclear localization sequences useful in the invention include sequences which resemble the short basic NLS of the SV40 T antigen described hereinabove; the bipartite NLS of nucleoplasmin the ribonucleoprotein sequence Al, the small nuclear ribonucleoprotein sequence U1A, and human T-lymphotrophic vims- 1 Tax protein; the HIV matrix protein NLS; and the nuclear translocation components impo ⁇ in/hSRPl and Ran/TC4.
  • a nuclear localization consensus sequence KXX(K/R) flanked by Pro or Ala Robotts (1989) Biochem. Biophys. Acta. 1008.
  • the invention thus also encompasses a nucleic acid condensing peptide which is linked to a non-basic nuclear localization sequence whose natural function is the transport of nucleoprotein.
  • a nucleic acid condensing peptide which is linked to a non-basic nuclear localization sequence whose natural function is the transport of nucleoprotein.
  • Such sequences are known in viruses, e.g., influenza nucleoprotein(Davey et al. Cell 40, 667 (1985)), HTV MA protein (Gallay et al., Cell 80, 379 (1995) .
  • Nucleoprotein transport sequences also occur in proteins which transport ribonucleoprotein complexes, e.g., hnRNP Al protein (Siomi and Dreyfuss J. Cell. Biol. 129. 551 (1995)) .
  • NBC2 nucleic acid condensing peptide linked to the M9 nuclear localization sequence of hnRNP
  • a nuclear localization sequence may be covalently linked to a nucleic acid condensing peptide of the invention as a functional group, or it may be present within the sequence of the nucleic acid condensing protein via peptide bond linkage as a fusion peptide.
  • Nucldc add condensing sequences especially useful in the invention comprise not only a nucldc add condensing domain, but also a nuclear localization sequence which directs transport of the vehicle to the nucleus.
  • nucleic acid condensing peptides are NBCl and NBC2, described herein, which include the SV40 T antigen nuclear localization sequence within the peptide sequence, as shown in Examples 1 and 2.
  • Example 10 shows that higher transfection activity is associated with use of NBC2 as a condensing peptide over NBC5.
  • NBC5 has the same stmcture as NBC2 except that the NLS has been inactivated by reversing the amino acid sequence of this part of the sequence. It is within the scope of the invention to combine peptides having different sequences, e.g., NBC2 (containing a nuclear localization sequence) and NBC9 (no nuclear localization sequence)
  • a nucleic acid condensing peptide of the invention which is conjugated to a functional group may be so conjugated via a bond which is stable enough to allow association of the peptide and the functional group to the cell surface and into the cell, if desired.
  • the following types of linkages are contemplated according to the invention.
  • bond stability is not an important variable (e.g. polymerization of the basic polymer unit) then a stable linkage is prefened.
  • Prefened stable bonds are amide, thioether and oxime.
  • NBC refers to a nucleic acid condensing peptide.
  • Amide linkages may be formed by reduction of Schiff base bonds formed by reaction of aldehydes and amino compounds. An example of this synthesis is given in Example 6.
  • the cytosol of mammalian cells is kept under net reducing conditions by the synthesis of reduced glutathione. Disulfide bridges, therefore, are cleaved upon absorption of molecules into the cytosol. This linkage is therefore useful for the coupling of peptides to the synthetic vims like particle whose function is not necessary after the particle has entered the cell (for example, a targeting protein or a fusogenic peptide) Disulfide linkages are readily synthesized by employing thiol residues activated with the 2-pyridyl group (Carisson et al., Biochem. J. 173. 723-737 (1978)).
  • the synthetic vims particle may comprise either a linear or branch chain polymer described in general terms by the formulae: LINEAR POLYMER: ⁇ P h ]
  • P is a protein or peptide or other chemical substance and b is 1-20, where P belongs to one of the following groups:
  • X is a nucleic acid binding component comprising an amino acid sequence or other biological sequence containing monomer units which are capable of binding to nucleic acid, and preferably containing at least one nuclear localization sequence, where a is 1-5;
  • L is a linker sequence comprising an amide, hydrazone.reduced hydrazone, disulfide, thioether or disulfide bond.
  • the linker sequence may be a lipid or polyethylene glycol.
  • Y is (-CH j ),, where t is 1-6, and preferably 1-5;
  • Synthetic vims like particles of the invention will possess an overall (net) charge as follows. Synthetic vims like particles which are designed so as to target a particular cell type, and therefore contain a targeting Iigand, will possess an overall charge in the range of 0.5 - 3.0, more particularly in the range of 0.5 - 2.0, and optimally in the range of 0.8 - 1.2. Particles which do not target a particular cell type, but are designed so as to transfect a broad range of cell types will possess an overall charge in the range of 0.5 - 5.0, more particularly in the range of 1.0 - 3.0, and optimally in the range of 1.3 - 3.0.
  • the overall charge (i.e., the balance of positive and negative charge species) of the synthetic vims like particle are determined as follows
  • Example 4 The nucleic acid condensing activity of peptides having different charges and the transfection effidendes of synthetic vims like particles having different charge ratios are presented in Example 4. Transfection efficiencies of particles containing varying amounts of ratios of positively/negatively charged residues are presented in Example 7. Highly efficient transfection may be obtained using particles of the invention which do not contain a targeting Iigand and are therefore untargeted with respect to a specific cell type. Such particles are highly efficient with respect to transfection where the ratio of positive/negative charges is greater than 1.25. 2) Overall Size
  • the size of a vims like particle fall within the range of 5nm to 500nm. It has been found that the efficiency of uptake of the particle by a cell dramatically decreases when the panicle size is greater than 500nm. This is likely due to the size of the endosomal pores in a given cell type. Particle size is measured by lasar light scattering or atomic force microscopy, or electron microscopy. Therefore, the size of a particle of the invention will vary depending upon the cell type and the size of endosomal pores in a given cell type.
  • a synthetic vims like particle according to the invention will have a ratio of the number of peptide/the number of nucleic acid molecules in a particle that is within the range of 10/1 to 1,000,000/1. This ratio will depend upon the relative sizes of the peptide and nucleic acid molecules, the degree of condensing activity of the peptide, and the degree of condensation that the nucleic acid attains. More particularly, the range will be 100/1 -10,000/1. For example, for NBC2 in combination with an 8kb vector, a useful ratio for untargeted delivery of the vector to cells is approximately 5000.1 (relative numbers of molecules). For NBC2 conjugated to insulin in combination with an 8kb vector, a useful ratio for targeted delivery of the vector to cells is approximately 1000:1.
  • the ratio of peptide/oligonucleotide is in the range of 0.1-10.0 and is preferably 0.5-1.0.
  • a synthetic vims like particle of the invention is formulated such that the nucleic acid and the peptide preparation are prepared in equal volumes of the same buffer.
  • the nucleic acid is agitated while the condensing peptide preparation is added at the rate of 0.1 volume per minute.
  • the complex is left at room temperature for at least 30 minutes prior to addition to the target cells and can be stored at 4°C.
  • the particle is centrifliged to remove any aggregated material and then assayed for gene transfer.
  • the nucleic acid/peptide complex is filtered through a sterile 0.2 ⁇ filter, e.g., a hydrophilic nylon membrane filter. Filtration of synthetic vims-like particles described herein does not significantly reduce yield, and in fact often results in 100% recovery of the particles.
  • nucleic acid condensing peptides useful in the invention are disclosed in detail herein.
  • a gel retardation assay may be performed which assesses the ability of the peptides present in the particle to condense nucleic acid. The gel retardation assay is performed as follows.
  • a concentration of nucleic acid is selected, for example 20, 30, or 40 ⁇ g/ml and possibly 50, 60, 70, or 100 ⁇ g/ml, and prepared in a low salt buffer, e.g., 150 mM NaCl.
  • the required amount of DNA is made up to 20 ⁇ g/ml in 150 mM NaCl; 25 mM HEPES, pH 7.4, or in 0.6 M NaCl; 25 mM HEPES, pH 7.4 and aliquoted between wells on a multiwell plate.
  • the amount of conjugate or peptide required to give positive charge:phosphate ratios of between 0.1 and 5.0 is calculated. This is made up to an equal volume to the DNA aliquots (0.05-0.5 ml) in either 150 mM sodium chloride; 25 mM HEPES, pH 7.4 or 0.6 M sodium chloride; 25 mM HEPES, pH 7 4.
  • the plate containing the DNA is placed on a plate shaker and shaken while the conjugate or peptide is added at a rate of 0.1 volume per minute. After addition of the condensing peptide is complete, the solution is incubated at room temperature for at least 30 minutes A sample for each positive charge:phosphate ratio is subjected to electrophoresis on an agarose gel The gel is stained with ethidium bromide and visualized under UV light. Condensed DNA remains in the well of the gel and does not migrate in the electric field.
  • Synthetic virus like particles are assayed for their ability to transfer genes into peripheral blood cells.
  • the plasmid DNA contains a marker gene for firefly luciferase.
  • the plasmid contains a gene whose expression will have a beneficial therapeutic effect.
  • the particle is incubated with the blood cells and the mbcture may be subject to electroporation if desired. After a further incubation, the cells are lysed and are assayed for gene expression.
  • luciferase reporter luciferin and ATP are added to lysed cells and the light emitted is measured with a luminometer.
  • Cells are harvested on the day of assay by centrifugation at 1200 rpm for 5 min at room temperature.
  • the cell pellet is resuspended in phosphate buffered saline and re-centrifuged. This operation is performed twice.
  • the cell pellet is then suspended in RPMI 1640 (Gibco Ltd.) to make up a suspension of 2.7 X 10 6 cells per ml.
  • the cells are then aliquoted into tubes and 0.75 ml of RPMI medium added, followed by 0.04-0.08 ml of 1 mM fusogenic peptide or more preferably 100 mM chloroquine and finally 0.25 ml of DNA-complex solution.
  • the transfection is then allowed to proceed by incubating the cells at 37°C for 4 h. After this time, the cells are harvested by centrifugation at 2000 rpm. The cells are then suspended in 1 ml of RPMI and re-centrifuged. Finally, the cells are suspended in 0.5 ml RPMI containing 10% foetal bovine serum. At this stage, if necessary, the cells are electroporated at 300 V and 250 ⁇ F using conventional electroporation
  • Each 0.5 ml of transfected cell suspension is transfened to a well of a 12 well plastic culture plate containing 1.5 ml of RPMI 10% FBS.
  • the original transfection tube is rinsed with a further 1 ml of medium and the wash transferred to the culture dish making a final volume of 3 ml
  • the culture plate is then incubated at 37°C for 24-72 h in an atmosphere of 5% CO 2 .
  • the contents of each well in the culture dish are transfened to centrifuge tubes and the cells collected by centrifugation at 13,000 rpm
  • the pellet is resuspended in 0.12 ml of Lysis Buffer (100 mM sodium phosphate, pH 7.8, 8 mM MgCl 1 mM EDTA; 1% Triton X-100 and 15% glycerol) and agitated with a pipette.
  • the lysate is centrifuged at 13,000 rpm for 1 minute and the supernatant collected. 80 ⁇ l of the supernatant are transferred to a luminometer tube.
  • the luciferase activity is then assayed using a Berthold Lumat L9501 luminometer.
  • the assay buffer used is Lysis buffer containing 10 mM Ludferin and 100 mM ATP. Light produced by the ludferase is integrated over 4 sec and is described as relative light units (RLU) The data are converted to RLU/ml of lysate, RLU/cell or RLU/mg protein (protein concentration of the lysate having been determined in this case by the BioRAD Lowry assay.
  • RLU relative light units
  • the ratio of groups present in the vims like particle may be preselected based on stoichiometric addition to the formulation of peptides containing the functional groups.
  • a selected functional group is a lipid
  • the amount of lipid present in the particle can be varied via stoichiometric addition of a lipid-conjugated peptide to the particle formulation
  • the stoichiometry of particle formulation may be varied by combining peptide preparations wherein one or more functional groups are present on a single peptide or where a single functional group is present on a single peptide
  • a peptide preparation may be added to a particle forming mixture which delivers a ratio of 1/1 of the two functional groups present on the single peptide, or which delivers a ratio of 1/1/1 or 1/2/1 (etc ) of three functional groups present on a single peptide
  • a second preparation may be added to the particle forming mixture which delivers a second ratio of, e.g , third/fourth/fifth fun ⁇ ional groups to the mixture
  • a second ratio of, e.g , third/fourth/fifth fun ⁇ ional groups to the mixture
  • the skilled artisan will have preselected the relative ratios of these functional groups
  • each peptide contains a single functional group
  • the amount of each peptide added to the particle forming mixture will determine the stoichiometry of addition of the functional group that is conjugated to the peptide.
  • the DNA may be condensed with a selected stoichiometry of condensing peptide, incubated for about 30 min, and then a second peptide containing a functional group is incubated with the condensed complex
  • Synthetic Vims-like Particles of the invention may be treated with a neutral hydrophilic polymer in order to form a more stable, smaller particle, and to increase transfection efficiency.
  • a neutral hydrophilic polymer will fall within the molecular weight range 1000 - 100,000; preferably within the molecular weight range 1000 - 50,000; and most preferably within the range 5000 - 10,000.
  • Such molecules are most useful according to the invention in a concentration in a given formulation within the range 0.5% - 10.0%; preferably within the range 0.5% - 5.5%; and most preferably within the range of 1% - 2%
  • a neutral hydrophilic polymer is used in this aspect of the invention according to the following procedure for formulating a synthetic vims like particle.
  • a synthetic vims like particle is prepared by combining a selected condensing peptide with a selected amount of neutral hydrophilic polymer. If desired, a physiological (i.e., isotonic) level (e.g., 0.15M) of salt is included at this step of the formulation. Nucleic acid is then added to the mixture, and the combination allowed to incubate at room temperature for at least 1 hour. The vims like particles will have assembled during this time. However, if desired, the mixture may be incubated for as long as 24-48 hours, or stored at a cool temperature (4C) overnight.
  • a physiological (i.e., isotonic) level e.g. 0.15M
  • Nucleic acid is then added to the mixture, and the combination allowed to incubate at room temperature for at least 1 hour.
  • the vims like particles will have assembled during this time. However, if desired, the mixture may be incubated for as long as 24-48 hours, or stored at a cool temperature (4C) overnight.
  • the amount of neutral hydrophilic polymer and salt added to the mixture during particle formulation may be determined as described in Example 12, and also using guidance provided in examples 5, 7, 13 and 15, and generally as follows
  • the particles may be prepared by combining 2 ⁇ g condensing peptide per ⁇ g DNA, at a DNA concentration of 100 ⁇ g/ml Different concentrations of neutral hydrophilic polymer are chosen for testing, each at a selected salt concentration
  • concentrations of neutral hydrophilic polymer to be tested may be on the order of 0.5%, 1.0%, 1.5%, 2.0%, 4.0% and 5.0%
  • the selected salt concentrations may be 0.2M, 0.4M, 0.6M, 0.8M and 1.0M.
  • the peptide is combined with the neutral hydrophilic polymer and salt in 100 ⁇ l of 25 mM HEPES buffer pH 7.4. 10 ⁇ g nucleic aicd is added in a small volume (several ⁇ l).
  • a prefened neutral hydrophilic polymer according to the invention is PEG, at a prefened concentration of 1-2%.
  • a prefened salt is sodium chloride at a prefened concentration of approximately 0.4M
  • the amount of nucldc acid and salt to be used in particle formulation may be determined as follows. Several different concentrations of nucleic acid are selected, for example, DNA concentrations (100 ⁇ g/ml, 250 ⁇ g/ml, 500 ⁇ g/ml, 750 ⁇ g/ml and 1000 ⁇ g/ml), each at different salt concentrations (for example, 0.4M, 0.6M and 0.8M NaCl). Briefly, 20 ⁇ g of peptide is mixed with a neutral hydrophilic polymer, for example, 2% PEG 8000, and the selected salt concentration in 25mM HEPES pH 7.4.
  • a neutral hydrophilic polymer for example, 2% PEG 8000
  • the concentration of peptide is such as to allow for a final peptide concentration of 200, 500, 1000, 1500, or 2000 ⁇ g/ml after addition of DNA and peptide. 10 ⁇ g DNA is then added to each mixture and mixed well. If desired, additional peptide, for example, in lipidated form, is added to the mixture. The mixtures are then left at room temperature for 1 hour before being stored at 4°C overnight. The particles are then assayed for transfection efficiency the following day.
  • the prefened nucleic acid concentration for particle assembly is 100 ⁇ g/ml or less.
  • 0.6M NaCl is optimal.
  • the transfection efficiency of particles of the invention decreases above 500 ⁇ g ml DNA during particle formulation.
  • the neutral hydrophilic polymer e.g., Polyethylene glycol (PEG, mw range 2000-15,000)
  • PEG Polyethylene glycol
  • the efficiency of transfection was found to be significantly increased by the presence of the neutral hydrophilic polymer.
  • the efficiency of transfection using a synthetic vims like particle of the invention is dramatically higher when transfection occurs in the presence of an agent which perturbs endosome function.
  • One such potent agent is the anti-malarial dmg chloroquine.
  • the action of chloroquine can be amplified by pre-incubation of the synthetic vims like particles in a solution containing chloroquine prior to treatment of the cells (presumably by adso ⁇ tion of this lipophilic molecule to the hydrophobic surface of the synthetic vims like particle). Increased transfection efficiency is observed when synthetic vims like particles prepared in the presence of lipidated peptide are pre-incubated with chloroquine before exposure to target cells.
  • Transfection efficiency may be further increased by increasing binding of chloroquine via elevation of the level of lipophilic substituents in the synthetic vims like particle.
  • the range of preincubation concentrations useful according to the invention are generally from 10 ⁇ M to 70mM.
  • the maximum amount of chloroquine administered with the synthetic vims like particle in vivo should not exceed 3.5 mg/kg body weight.
  • the final concentration of chloroquine after dilution from the formulation is in the range of 50-200 ⁇ M.
  • transfection efficiency is increased by extending the time period to which the target cells are exposed to the synthetic vims like particle in the presence of chloroquine This time period may be from 2 hours to as much as 24-48 hours, with the longer incubation times resulting in increased transfection efficiency in the presence of chloroquine.
  • Example 17 experiments are presented which demonstrated increased transfection efficiency using a synthetic vims like particle prepared according to a novel procedure which includes preincubation of the synthetic vims like particle with chloroquine.
  • High Salt Formulation of Synthetic Vims Like Particles of the Invention It has been discovered that increased transfection efficiency is obtained using a synthetic vims like particle which is prepared according to a novel procedure which includes the use of a high salt formulation. "High” salt is defined herein as being within the range of 0.5-1.0M salt, “low” salt is defined as being in the range of 0 1 -0.2M salt This procedure is as follows.
  • Nucleic acid is made up to 90-120 ⁇ g/ml in 25 mM HEPES buffer containing 0.6-1.0M sodium chloride.
  • Nucldc add condensing peptide is made up to an equivalent molarity in sodium chloride and the condensing peptide solution added to the nucleic acid solution with rapid agitation at a rate of 0.1 vol/min The mixture is then left for at least 30 minutes at 20° C and then incubated for 1-16h at 4° C prior to dilution to a concentration of 5 ⁇ g/ml nucleic acid in RPMI medium (optionally containing albumin lmg/ml, transferrin 50 ⁇ g/ml) and containing the endosome escape agents, fusogenic peptide (20-100 ⁇ M) or chloroquine (100-200 ⁇ M ).
  • Lipidated peptides useful according to the invention cannot be inco ⁇ orated in the initial high salt formulation solutions described above when the DNA concentration is above about 250- 300 ⁇ g/ml.
  • the synthetic vims like particles produced exhibit minimal biological activity. Therefore, where a lipidated peptide is used in the synthetic vims like particle, the particles must be formulated such that they retain biological activity.
  • High salt formulation of such panicles is performed as follows.
  • Lipidated peptides are incorporated into the RPMI dilution medium at a concentration of 0-2 ⁇ g/ ⁇ g DNA, (absolute concentration 0-10 ⁇ g ml) and the solution incubated for at least 30 minutes at 37° C prior to transfection
  • the synthetic vims like particle is formulated in high salt, as described above, and the synthetic vims like particle incubated overnight at 4°C before the lipidated peptide is added to the synthetic vims like particle from stock solution of lmg/ml lipidated peptide in 25 mM Hepes buffer containing 0.15 M sodium chloride.
  • Transfection effidendes of particles containing lipidated peptides, which particles are formulated in high salt are described in Example 7.
  • Nucldc add condensing peptides of the invention are synthesized so as to achieve a high degree of homogeneity in the peptide preparation, both in terms of the polydispersion index and the addition of functional groups.
  • a peptide preparation consists of a selected amino add sequence
  • care is taken during synthesis to ensure homogeneity of the heteropeptide sequence by ensuring complete coupling to the growing peptide chain.
  • Homogendty of the peptide preparation also is ensured by protecting potentially reactive amino acid side chains in the growing peptide. 606
  • N-Palrnityl de ⁇ vatives of the co ⁇ espondmg NBC peptides are called Lipl, L ⁇ p2 etc e.g The stmcture of L ⁇ p2 is
  • Cholesteryl derivatives are named after the corresponding NBC peptides, Choll. Choll2 etc e.g.
  • the structure of Choll2 (Chol 1 ⁇ -NBC12' (superscripts denote the site of de ⁇ vatizauon) is
  • lnsla BI -NBC14' (superscripts refer to linkage site on Iigand and peptide) has the following stmcture :
  • InsN B '-NBC14' has the following stmcture Insulin O
  • Insul ⁇ n B '-Chol ⁇ 1 -NBC14' l8 has the following stmcture
  • Man 4 Den2- NBC12' The stmcture of Mannosylated Lysine Dend ⁇ mer conjugated to NBC 12 (Man 4 Den2- NBC12') is:
  • Man 4 Den5-NBC12 and Man 4 Den6-NBC12 are analogues of Man 4 Den2 and Man 4 Den3 respectively and differ in stmcture by the linkage of the mannose residues to the Lysine dendrimer backbone. In these cases the sugar is linked through the hydroxyl group of N-terminal serine res i dues as in the case of Man 4 Den5-NBC12. Also there i s no cys-male i midophenyl butyrate linker between the dendrimer and the NBC pept i de The stmcture of Man4Den5-NBC12 i s
  • Peptides NBCl -14 were synthesised using a Biosearch 9050 plus Pepsynthesizer in extended synthesis cycle mode using Fmoc-Cys(Acm)-0-PEG-PS-Resin with dimethylformamide as solvent. Deprotection of the N-terminal Fmoc-group before each coupling was achieved using a solution of 20% piperidine in dimethylformamide(lmin at a high flow rate followed by 10 min at 3ml/m ⁇ n).
  • the amino acids were coupled in four fold excess using O-(lH-benzot ⁇ azo-l-yl)-tetramethyluronium tetrafluoroborate (TBTUyi-hydroxybenzot ⁇ azole and N-ethyldiisopropylamine as activating agents.
  • TBTUyi-hydroxybenzot ⁇ azole and N-ethyldiisopropylamine activating agents.
  • NBCl the coupling times started at 30 mm increasing by 15mins after every 15th amino acid during the synthesis to 1.25h. for the last 15 amino acids.
  • For all the other NBC- peptides. coupling times were set at lhour throughout the synthesis.
  • the following ammo acid de ⁇ vatives were used as approp ⁇ ate for the NBC: Fmoc-Ala-OH. Fmoc- Arg(Pbf)-OH.
  • Peptides were cleaved from the resin using J TFA water/phenol th ⁇ oan ⁇ sole/1.2- ethanedithiol (82.5: 5: 5: 2.5) mixture ( 10ml of mixture for every gram of resin to be cleaved). The resin was then removed by filtration and washed 3 umes with TFA. The combined filtrate and washings were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to give the cmde peptide.
  • Cmde peptides were dissolved in a small amount of 1 % acetic acid in water and applied to a Sephadex G25 (superfine) column of an appropriate size and eluted using the same 1 % Acetic acid solution.
  • the fractions containing peptide, as determined by analytical reverse phase hplc, were pooled and lyophilised. Further purification was achieved by preparative reverse phase hplc using a Dynamax 83-221-C column and a gradient of 5- 30% Acetonitrile(0.1 % TFA) in Water(0.1 % TFA) over 20 min.
  • the fractions containing peptide were pooled, the acetonitrile evaporated in vacuo , and lyophilised.
  • NBC2-NBC14 were purified by preparative rp hplc, desalted by elution on a Sephadex G25 superfine column ( 1.6x30cm) with I % acetic acid in water. The resulting fractions were analysed by reverse phase hplc, pooled and lyophilised as above.
  • the acetamidomethyl (Acm) thiol protecting group maybe removed using mercury (11) acetate in 30% acetic acid in water followed by precipitation of the mercury with 2-mercaptoethanol.
  • the resulting free thiol peptide can be purified using gel filtrauon to give the desired product.
  • M ⁇ is the weight average molecular weight and M n is the number average molecular weight.
  • the usual method for determining the weight average molecular weight of heterogeneous polymers such as poly-lysine is to use light sca ⁇ e ⁇ ng where the amount of light scattered depends on molecular size as well as the number of panicles In these cases the number average molecular weight is calculated from a col gauve prope ⁇ y of the polymer in soluuon such as viscosity since this property is dependent on the number of molecules of polymer per unit volume
  • Poly-L-Lysme 25000. Sigma Product P7890, Lot No 93H-5516 has a PDI of 1.2. This result is typical for the batches of poly-lysme used in many gene transfer expe ⁇ ments reported in the literature
  • Fig 2 shows the deconvoluted electrospray mass spectrum of peptide NBC9 (theoretical mass 4082.2).
  • An aqueous solution of lmg/ ml peptide was prepared and the sample diluted in a mixture of aeeton ⁇ trile:methoxyetha ⁇ ol:0.1% trifluoroacetic acid.
  • the experiment was performed on a VG Instmments Quattro II instrument fitted with a 41606
  • the instmment was calibrated with myoglobin and lO ⁇ l aliquots were injected directly into the instmment source.
  • the nucleic acid is made up to 20-400 ⁇ g/ml in the buffer (usually 0.15 M to 1.0 M
  • the required amount of conjugate or peptide is made up to an equal volume to the nucleic acid solution in the same buffer.
  • the DNA is agitated moderately while the condensing agent is added at the rate of 0.1 volume per minute.
  • the complex is left at room temperature for at least 30 minutes prior to adding to cells and can be stored at 4°C
  • Figure 3 shows the comparative gel retardation behavior of NBCl (31 positive charged groups), NBC7 ( 16 positively charged groups), NBC8 (13 positively charged groups), NBC9 (19 positive charged groups) and NBC10 (25 positively charged groups) 1 ⁇ g aliquots of plasmid RSVluc DNA (20 ⁇ g/ml in 150 mM NaCl in 25 mM
  • HEPES.pH 7.4 HEPES.pH 7.4 were aliquoted into a multi-well plate (50 ⁇ l volume). The amount of each of the peptides required to give positive charge: phosphate ratios of 0, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0. 2.5. 3.0. 3.5 and 4.0 was calculated and made up to
  • DNA was placed on a plate shaker and shaken whilst the peptide solution was added to the DNA at a rate of 5 ⁇ l per minute. After addition of the condensing agent is complete, the solution was incubated at room temperature for at least 30 minutes. A sample for each positive charge: phosphate ratio is electrophoresed on a standard 1% TAE agarose gel. The gel was stained with ethidium bromide and visualized under UV light. Condensed DNA remains in the well of the gel and does not migrate in the electric field. 606
  • Each gel shows the electrophoretic mobility of the same quantity of plasmid DNA after mixing with various ratios of each peptide (equal to 0, 0.15, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, and 4.0 equivalents of positive charge (Lysine +
  • the nucleic acid used is plasmid DNA contains a marker gene for firefly luciferase (pRSVLuc; de Wet J.R., Wood K.V.. DeLuca M., Helsinki D.R.. and Subramani S. (1987) Mol. Cell. Biol. vol. 7, pp 725-737).
  • pRSVLuc firefly luciferase
  • After incubation the cells are lysed and are assayed for gene expression.
  • the luciferase reporter gene lucife ⁇ n and ATP are added to lysed cells and the light erru ⁇ ed is measured with a luminometer.
  • Cell lines such as Jurkat and K562 grow in suspension and can be transfected and the level of transgene expression (luciferase activity) determined by the following method.
  • Cells are harvested on the day of assay by centrifugation at 1200 rpm for 5 min at room temperature.
  • the cell pellet is resuspended in phosphate buffered saline and re- centrifuged. This operation is performed twice.
  • the cell pellet is then suspended in RPMI 1640 (Gibco Ltd.) to make up a suspension of 2.7 x 10 6 cells per ml.
  • the cells are then aliquoted into tubes and 0.75 ml of RPMI medium added, followed by 0.04 - 0.08ml of lOOmM Chloroquine and finally 0.25ml of DNA-complex solution.
  • the transfection is then allowed to proceed by incubating the cells at 37°C for 4h. After this time the cells are harvested by centrifugation at 2000rpm.
  • the cells are then suspended in 1ml of RPMI medium and re-centrifuged. Finally the cells are suspended in 0.5ml RPMI medium containing 10% foetal bovine semm. Each 0.5ml of transfected cell 1606
  • the method can be modified for adherent cells such as the hepatic carcinoma cell line HepG2 Cells were plated 24h p ⁇ or to transfection in 6-well ussue culture plates at a density of 1-2 x 10 5 per well in complete medium (MEM (with Earle's salts) + 1% non- essential amino acids + 10% foetal calf sem ) Transfection was carried out by aspirating the culture medium and washing the cells with phosphate buffered saline.
  • MEM with Earle's salts
  • the cells were then harvested using trypsm to detach the cells. After addition of medium containing semm to inactivate the trypsin, cells were pelleted by centrifugation at 13,000 rpm. The cells were then suspended in 1ml of PBS and re-centrifuged. The pelle? was resuspended in 200 ⁇ l of Lysis Buffer (lOOmM sodium phosphate, pH 7.8; 8mM MgC12, ImM EDTA; 1% Triton X- I00;15% glycerol; 0.5mM PMSF and 1 mM Dithiothreitol) and agitated with a pipene.
  • Lysis Buffer lOOmM sodium phosphate, pH 7.8; 8mM MgC12, ImM EDTA; 1% Triton X- I00;15% glycerol; 0.5mM PMSF and 1 mM Dithiothreitol
  • the lysate is ceuuiftiged at 13,000 rpm for 1 minute and the supernatant collected. 80ml of the supernatant are transferred to a luminometer tube. The luciferase activity is then assayed using a Berthold Lumat L9501 luminometer.
  • the assay buffer used is Lysis buffer containing 0.0 ImM Luciferin and 0.04375mM ATP. Light produced by the luciferase is integrated over 4sec and is described as relative light units (RLU).
  • the data are converted to RLU / ml of lysate, RLU/cell or RLU/mg protein (protein concentration of the lysate having been determined in this case by the BioRAD Lowry assay.
  • FIG. 4 shows non-receptor mediated transfection by synthetic vims like particle prepared by mixing increasing amounts of NBCl and NBC2 with plasmid DNA.
  • Figures 4(a)-(c) show that high levels of transfection can be obtained by increasing the proportion of the nucleic acid condensing peptide in the formulation. The experiment was performed as follows. Stock solutions of NBCl and NBC2 were made up at concentrations of 2.5,
  • pRSVLuc DNA at a concentration of lOO ⁇ g/ml was condensed using peptide at a ratio of 2 ⁇ g/ ⁇ g plasmid
  • Figure 6a shows the results of a similar expe ⁇ ment.
  • AH conditions were identical to the previous example except thai in this case the DNA was co-condensed with 0.6 ⁇ gL ⁇ pl3
  • Figure 6b shows a similar compa ⁇ son of data using NBCl . NBC8. NBC9 and NBC 10 as condensing peptides
  • Cell targeung components such as monoclonal antibodies may be conjugated to a nucleic acid condensing peptide according to the invention.
  • the Carbohydrate groups present on the monoclonal antibody are oxidized using pe ⁇ odic acid to produce reacuve aldehyde groups (Jentoft and Dearborn J. Biol. Chemi. 254. 4359 ( 1979) ) .
  • the oxidized antibody is then reacted with ammo groups present on the NBC2 peptide. This results in the formation of an imine (Schiff Base) linkage between the antibody and the NBC polymer.
  • the imine linkage is very labile in water and must be reduced using sodium cyanoborohydride or sodium borohydride to give the stable amine.
  • Ant ⁇ -CD7 antibody 50 mg at 5 mg ml in 25 mM sodium acetate, pH 5.0 was oxidized by the addition of sodium pe ⁇ odate to 10 mM. The solution was left on ice, in the dark for lh. The oxidized antibody was desalted and the buffer exchanged to 25 rnM MES, pH 6.0 on a Sephadex G25 column (30 cm x 2.5 cm i.d.) . The amount of antibody recovered from the column was determined by measuring the absorbance at 280 nm (the absorbance of a 1 mg/ml solution of antibody at 280nm is 1.33). A 5 times molar excess of NBCl was added and the solution was left at room temperature for 1 h.
  • the unmodified antibody does not bind to the column and is washed through.
  • the antibody conjugated to NBCl binds to the column and is eluted early in the gradient (Fig. 7) .
  • the conjugate peak eluted at approximately 1M NaCl. 1606
  • NBC2 A total of 250 nmol NBC2 (as determined using an Ellman's test ) were added to the antibody-PDPH and the solution was left for 16 h at room temperature.
  • concentration of NaCl was adjusted to 0.15 M before the ant ⁇ body-NBC2 conjugate was purified by cation exchange on SP Sepharose using NaCl gradient of 0.15- 3 M. The separation obtained is shown in Fig. 4.
  • the cmde conjugate was applied to the column [S-Sepharose Fast Flow] on 0.15 M NaCl and eluted with a 0.15- 3 M linear gradient of NaCl.
  • the conjugate peak eluted at approximately 1M NaCl.
  • the protein peak was pooled and dialyzed against 0.02 M HEPES buffer. 0.15 M sodium chloride buffer. pH 7.2.
  • the derivatized antibody was reduced with cysteine and purified by gel filtration using 25 mM HEPES, 0.5 M NaCl, pH 7.9, as mnning buffer. The fractions were pooled and the concentration of thiol groups determined using an Ellman's test. A 2 606
  • the peptide was synthesized using a Millipore 9050 plus peptide synthesizer equipped with enhanced counter-ion distribution monitoring to control the coupling time. Fmoc-Gly-O-Resin was used. After deprotection of the Fmoc group, using 20%, piperidine in dimethylformamide. the subsequent amino acids are coupled in four-fold excess using 0.6 M N.N'-diisopropylcarbodiimide with 0.025% Quinoline Yellow in dimethylformamide and 0.6 M 1 -hydroxybenzotriazole with 1 mM diisopropylethylam e in dimethylformamide as activating agents.
  • the N-termmal Fmoc group was removed as described above and the peptide was cleaved from the resin using 95:5 TFA/water mixture. Purification of the peptide by gel filtration on Sephadex G25 followed by preparative reverse phase hplc gave the desired product.
  • the peptide may then be treated with 2-bromoacetic acid N- hydroxysuccinimidyl ester water to give the N-(2-bromoacetyl)-peptide.
  • 2-bromoacetic acid N- hydroxysuccinimidyl ester water to give the N-(2-bromoacetyl)-peptide.
  • reaction of a solution of the peptide in water with a large excess of hydrazine in the presence of EDC-I to yield the N- -bromoacetyl) -penta-glutaryl-hydrazide derivative of the peptide.
  • the concentration of NaCl- was adjusted to 0.15 M before the clustered antibody-NBC2 conjugate was purified by cation-exchange on SP Sepharose using a NaCl gradient of 0.15- 3 M
  • the protein peak was pooled and the pool ste ⁇ le filtered and the conjugate stored at 4°C
  • Figs 9- 14 These figures are histograms showing the relauve activity of luciferase in lysates de ⁇ ved from cells transfected with plasmid DNA containing the luciferase gene under the contfol of the RSV promoter There is no endogenous luciferase acuvity in any of the cell lines or p ⁇ mary cells used
  • Fig 9 shows that gene transfe ⁇ to myeloid cells can be targeted using an ant ⁇ -CD33 complex in which the DNA is condensed with ant ⁇ -CD33-NBCl con j ugate in dose dependent manner
  • the ant ⁇ -CD33-NBCl conjugate was synthesized as described in Example 6 1 1 1
  • This expe ⁇ ment shows the effect of complex concentration on gene transfer At 2.5 ⁇ g/ml DNA complexed with unconjugated NBC2 no gene transfer was observed As the amount of complex added to the cells is increased the level of luciferase (transfection) increases to a maximum of 1 -2 ⁇ g/DNA complex/1 x 10 6 cells
  • Fig 10 shows that the overall loading rauo of the plasmid with coniugate is important for activiry with lower activity associated with complex prepared with an excess of conjugate Ant ⁇ -CD33-NBCI complex was prepared such that the ratio of conjugate to DNA remained constani but the amount of unconjugated NBC2 was varied to give total cat ⁇
  • Cell targeung is specific, as shown by the observauon that cells treated with free and compe ⁇ ng unconjugated ant ⁇ -CD33 antibody have reduced levels of transfection when exposed to complexes formed using ant ⁇ -CD33-NBCl (prepared as described in
  • Example 6.1.1.1 in the standard assay system (Fig. 11), and by the fact that no gene transfer is observed when cells are exposed to DNA condensed with unconjugated
  • Fig. 12 shows that the quantity of antibody within the complex can be optimized to increase transfection efficiency.
  • Complexes were prepared as described in Example 4 with various proportions of anti-CD7-NBCl conjugate (prepared as described in Example 6.1.1.1 ) and unconjugated NBC2. The final quantities were adjusted so that DNA condensation as determined by the gel retardation assay did not vary.
  • Fig. 12 shows that when 25% of the condensation is provided by interaction between the nucleic acid and anti-CD7/peptide conjugate and 75% by free NBC2 maximal transleclion efficiency is obtained
  • Figure 13 shows the time course of expression.
  • Cells were transfected with anti-CD7 conjugate prepared as described in Example 6.1.1.1 and assayed as described in Example 4 except that the cells were grown for increasing time periods before lysis and analysis of luciferase expression.
  • Insulin was chemically modified according to previously described methods (pp.43-44 of Offord. R.E.(1980) 'Semisynthetic Proteins " 235pp.. Wiley, Chichester and Ne York), with slight modifications B ⁇ cfh .
  • lOOmg Zn-free insulin were dissolved in 1 l of 1M sodium hydrogen carbonate, diluted with 4mL dimethylformamide and reacted with an equimolar amount (relative to peptide amino groups) of methylsulphonyloxycarbonic acid N-hydroxysuccinimide ester.
  • the reacuon medium was acidified and diluted with 0 1 %TFA, and the de ⁇ vatized insulin isolated by semj- preparative HPLC on a C8 column equilibrated in 0 1% TFA, using a 35-45% gradient (same eluants as desc ⁇ bed above) over 20mm
  • the methylsulphonyloxycarbonyl groups were then cleaved under standard conditions and the mate ⁇ al repu ⁇ fied on the C8 column using a 35-40% gradient over 20rwns The final compound. Boc-AoA- insulin.
  • the Cys-protected peptide was oxidized as follows The pepude was dissolved in 50 mM lrrudazole (Cl), pH 6 9 at a concentration of 10 mg/mL. and 0.2M methionme m water was added (as an anti-oxidant scavenger), a 10-fold molar excess over peptide Then 50mM sodium penodate was added to a five-fold molar excess over peptide.
  • Periodate oxidized NBC 14 was prepared as described in Example 6.2.1.2. 4mg of this material were mixed with 4mg of des-insulin derivative in 400 ⁇ l of 0.1 M sodium acetate buffer containing 20% acetonitrile, pH 3.6. After 15h incubation at room temperature the conjugate was isolated by preparative hplc using a 1cm diameter C8 column and a 30-35 % gradient of acetonitrile solvent (using Solvent A and Solvent B as described above). The recovered material (3.2mg) was characterized by electrospray mass spectrometry (calculated mass 7465 / observed mass 7462).
  • N-succ ⁇ n ⁇ m ⁇ dyI 3-(2-py ⁇ dyld ⁇ th ⁇ o) propionate (SPDP) was reacted separately with both insulin and commercial polv-L-Lysine
  • the two de ⁇ vatives were mixed after reducuon of the 2-py ⁇ dyl disulphide group in modified poly-L-lysine.
  • the insulin receptor targeted synthetic vims like particles were assembled using pRSVLuc plasmid DNA and Insul ⁇ n-NBC14 conjugates as described in Example 4.
  • the cells were then harvested using trypsin to detach the cells. After addition of medium containing semm to inacti'. ate the trypsin, cells were pelleted by centrifugation at 13,000 rpm. The cells were then suspended in 1ml of PBS and re-centrifuged. The pellet was resuspended in 200 ⁇ l of lysis buffer (lOOmM sodium phosphate, pH 7.8: 8mM MgC12, ImM EDTA; 1 % Triton X-100;15% glycerol; 0.5mM PMSF and 1 /41606
  • the assay buffer used is Lysis buffer containing 0.0 ImM Luciferin and 0.04375mM ATP.
  • RLU relative light units
  • Figure 14 shows the relative transfection potency of defined insulin-NBC14 conjugates and a defined conjugate of a non-receptor binding insulin analogue (des B2 11 Insulin- NBC14) and insulin conjugates of poly-L-Lysine prepared by the approach of Bimstiel ei.al. loc cu These results show that targeting is specific and depends on the presence of an insulin conjugate which is able to recognize the receptor (InsI-NBC14 is active whilst des BJ " lnsul ⁇ n-NBC I4 is totally inactive).
  • Insulin- NBC 1 based gene delivery is at least 2 orders of magnitude higher than the best analogous insulin-poly-L-Lysine based delivery.
  • the greatly reduced activity of the insulin-poly-L-lysine is due to largely to the poor transfection properties of poly-L- ivsine.
  • Insulin has 3 amino groups available for conjugation.
  • Giycosyl moieties may be clustered by the use of branched carbohydrates of natural or synthetic origin. Alternatively these groups may be clustered by modifying a branched amino acid backbone (dend ⁇ mer)
  • glycosylated dendrimeric moieties described in this section are based on a generic design of a Iigand that, when conjugated to an NBC peptide, will give optimal binding of the peptide to either the hepatocyte asialoglycoprotein receptor (ASGPR) or the macrophage mannose receptor (MMR).
  • ASGPR hepatocyte asialoglycoprotein receptor
  • MMR macrophage mannose receptor
  • the type of monosaccharide used in the synthesis of the Iigand will depend on the receptor of interest.
  • the design of the Iigand is based on a consideration of the binding requirements of the ASGPR and the MMR, as it is these receptors that are of p ⁇ mar interest for use in gene targeting.
  • the literatv ⁇ states that ligands which present several (i.e three or more) monosaccharide units clustered together with their non-reducing ends exposed generally 1606
  • the 2nd generation lysine dend ⁇ mer [Dcn2 (Lys),-Gly-Gly-Tyr-Cys] was synthesised using a Millipore 9050 Plus Pepsynthesiser mnning in extended cycle mode (1 h couplings) with TBTU / N-cthyldiisopropylamine activation and deprotection with 20 % Piperidine in dimethylformamide Starting from Fmoc-Cys(Trt)-O-PEG-PS resin, and with a four-old excess of activated ammo acid to free amine group, the peptide was synthesised sequentially using the following amino acid derivatives; Fmoc-
  • the peptide was cleaved from the dried resin using reagent K (TFA, water, phenol,thioanisole,l,2-ethanedithiol; 82.5:5:5:5:2.5) and purified by ion-exchange (SP- sepharose)and reverse phase hplc. 06
  • reagent K water, phenol,thioanisole,l,2-ethanedithiol; 82.5:5:5:5:2.5
  • SP- sepharose reverse phase hplc. 06
  • the thiol of the mannosylated peptide was reduced by adding 20 mg solid dithiothreitol.
  • the peptide was then purified by high resolution gel filtration.
  • the mannosylated peptide was determined to possess, on average, between 3 and 4 mannose groups per peptide molecule.
  • a maleimide function was introduced into NBC 12 by addition of 4.3 mg (12.2 ⁇ mol)
  • Man wellDen5-NBC12 is a mannosylated dend ⁇ mer de ⁇ vative of NBC- 12. Similar in stmcture of Man 4 Den2-NBC1 '. In this case however the mannosyl side chains are introduced using solid phase peptide synthesis coupling methods with the tetra-o-acetyl- mannose- ⁇ -Fmoc-Se ⁇ ne de ⁇ vative
  • the peptide dend ⁇ mers were synthesised using a Millipore 9050 Plus Pepsynthesizer ⁇ described above except that Fmoc-N 1 -/er/-butoxycarbonyl lysine was replaced by di- Fmoc-lvsine.
  • glycopeptides were cleaved from the resin by adding an excess of 95% TFA for 2 h at room temperature.
  • the glycopeptide was precipitated in 10 volumes of ether, the ether discarded after centrifugation and purified by hplc.
  • the glycopeptide, Man4De ⁇ 5 was coupled to NBC 12 via a maleimide-hydrazide bifunctional crosslinker, as described above.
  • the glycopeptide was synthesised with a C-ter ⁇ unal hydrazide group by using a hydroxymethylbenzoic acid (HMBA) resin linker and cleavage method, as described in "synthesis of Man4Den4".
  • HMBA hydroxymethylbenzoic acid
  • Such hydrazide derivatives can be used to couple the Man peptide directly to oxidised NBC 12, as described for the synthesis of Man4Den4-NBC12.
  • Palmitic acid N-hydroxysuccinimide ester (0.5g per gram of resin) was added to a suspension of N-termmal ammo deprotected resin bound peptide (side chain protected)- O-PEG-PS resin in methanol/chloroform (1 :4; 5ml per gram of resin synthesized as described in Example 2) The reaction was shaken for 48h. at room temperature then the resin was filtered off. washed 3 times with chloroform, washed with methanol and dried in vacuo
  • the peptide was cleaved from the resin using a TFA/water/phenol thioanisole/1.2- ethanedithiol (82.5: 5: 5: 5: 2.5) mixture (lOmJ of mixture for every gram of resin to be cleaved)
  • the resin was then removed by filtration and washed 3 times with anhydrous t ⁇ fluoroacetic acid.
  • the combined filtrate and washings were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to give the cmde peptide.
  • the cmde peptide was dissolved in a small amount of 25mM HEPES pH 7.4 and applied to an SP-Sepharose fast flow , column of an appropriate size and eluted using a gradient of 0-2M NaCl in 25mM HEPES pH 7.4 over 20 column volumes.
  • the fractions containing the peptide as shown by analytical reverse phase hplc, were pooled and applied directly to a preparative reverse phase hplc (Dynamax 83-221-C column) and eluted using an approp ⁇ ate gradient of acetonitrile (0.1% TFA) in water (0.1 % TFA) (typically 30-50% acetonitrile over 20 min).
  • Palmitic acid N-hydroxysuccinimide ester (25.7mg) was added to a stirred solution of
  • Lipid containing complexes may be assembled as described in Example 4.
  • the lipo- pepude may be mixed with the NBC condensing peptide solution prior to the addition of the peptide mixture to the DNA solution Alternatively the lipopeptide may be added to the complex after condensation with the NBC. before or after the dilution step p ⁇ or to addition to the cells. In these cases, the complexes should be incubated overnight before addition of the lipopeptide
  • Fig. 17 shows the effect of N-palm ⁇ toyl-NBC2 on transfection efficiency of a an anti-CD7-NBC2 conjugate.
  • Complexes were made up as described in Example 4. The DNA was first treated with conjugate such that 50% of the material was condensed as determined using the gel retardation assay. Various mixtures of unconjugated NBC 1 and N-Palmityl-NBCl were addedsuch that complete condensation was obtained.
  • Fig. 18 shows data similar to those shown in Fig 17 except the effect of N-palmitoyl-NBCl on transfection efficiency of a an anti-CD33-NBC2 conjugate is shown.
  • Figure 19 shows that at an optimal ratio of L ⁇ p2 to NBC2 can boost transfection activity by > 2 orders of magnitude.
  • Figure 20 shows that this effect is a common property of de ⁇ vatized peptides of low polydispersity. These data also shows that peptides which have poor relative transfection efficiency (e.g.
  • Fig 22 shows the differential transfection levels obtained with two different cell lines K562 and HepG2 and the same formulation L ⁇ p2 in NBC2. In the case of K562 cells little boost in activity was observed whereas in the case of HepG2 the optimal ratio was
  • the optimum ratio for Jurkat cells is 0.13-0.6 ⁇ g peptide/ ⁇ g DNA.
  • FIG. 23 shows the effect on transfection efficiency of pre-incubating the synthetic vims like particle with varying amounts of human plasma.
  • the presence of Lip2 in the formulation clearly confers added resistance to this degradation.
  • the NBC2 synthetic vims like particles were prepared essentially as described above using a DNA formulation of 1.5 ⁇ g NBC2 and were diluted into 25 mM Hepes, 0.85 M sodium chloride, pH 7.4 to a concentration of 10 ⁇ g DN A/ml.
  • Lip2 synthetic vims like particles were prepared by mixing 1.5 ⁇ g Lip2/ ⁇ g DNA in 25 mM Hepes, 0.85 M sodium chloride, pH 7.4.
  • the synthetic virus like particles were diluted into an equal volume of dilution/transfection medium containing 50%, 25%. 10% and 0% freshly prepared human plasma and 100 ⁇ M chloroquine.
  • the solutions were then mixed with 1 x I0 6 Jurkat cells in RPMI containing 120 ⁇ M chloroquine. After 4h the cells were centrifuged, the medium removed and the cells resuspended in 2.5 ml of RPMI medium containing 10% foetal calf semm. After 24h the cells were collected, washed in 25 mM Hepes, 0.85 M sodium chloride, pH 7.4, lysed and the level of luciferase expression determined as described above.
  • the formulations were prepared as follows:
  • the complexes were assayed for the efficiency of luciferase gene transfer using Jurkat cells as described in Example 4 The results are shown in Fig 24.
  • Combinations of lipid and targeting Iigand may also be attached to the nucleic acid binding peptide.
  • This conjugate has a similar stmcture to Man 4 Den2-NBC12' except the mannosylated lysine dendrimer Iigand is separated from the nucleic acid binding peptide by an eleven carbon hydrocarbon spacer.
  • the lysine dendrimer Den, (Lys),-Gly-Gly-Tyr- aminoundecanoyl-Cys- was synthesised using a Millipore 9050 Plus Pepsynthesiser mnning in extended cycle mode ( 1 h couplings) with TBTU / N-ethyld ⁇ sopropylamine activation and deprotecuon with 20% piperidine in dimethylformamide.
  • the peptide was synthesised sequentially using the following am o acid de ⁇ vatives; Fmoc- amino-undecanoic acid, Fmoc-0(/ rr-butyl)-tyrosine, Fmoc -glycine, di-Fmoc-lysine and Fmoc-N'-r -butoxycarbonyl lysine. After the final coupling had finished the N- terminal Fmoc groups were removed using 20% Piperidine in dimethylformamide.
  • the peptide was cleaved from the dried resin using reagent K (TFA, water, ⁇ henol.th ⁇ oan ⁇ sole,1.2-ethaned ⁇ th ⁇ ol. 82.5:5:5:5.2.5) and purified by ion-exchange (SP- scpharosciand reverse phase hplc
  • the thiol of the mannosylated peptide was reduced by adding 20 mg solid dithiothreitol.
  • the peptide was then purified by high resolution gel filtration.
  • the mannosylated peptide was determined to possess, on average, between 3 and 4 mannose groups per peptide molecule.
  • the glycosylated lipid dendrimer was conjugated to NBC 12 in an analagous way to that described in 6.3.2.1. 7.3.2 Insulin -NBC14'- 20 -Cholesterol Conjugate
  • a functional group is conjugated to a peptide of the invention as follows.
  • One representative functional group is a fusogenic peptide.
  • a fusogenic pepude (FPI3) derived from the Influenza HA may have the sequence:
  • the peptide was synthesized using a Millipore 9050 plus peptide synthesizer in extended synthesis cycle mode ( 1 -hour couplings).
  • Fmoc-Cys(Acm)-O-PEG-PS resin Persepuve Biosystems Ltd.
  • the subsequent ammo acids were coupled in four-fold excess using 0-(lH-benzounazol-i-yl)-tetramethyluronium tetrafluoroborate (TBTUyi-hydroxybenzot ⁇ azole and N-ethyl-diisopropylamine as acuvaung agents.
  • the N-terminal Fmoc group was removed as described above to give the free N-terminal amino side chain protected, peptide bound to the resin. This was cleaved from the resin using a TFA/water/phenol/- th ⁇ oan ⁇ sole/1.3-ethaned ⁇ th ⁇ ol (82.5:5:5:5.2.5 ) mixture. Following precipitauon with ether and centrifugation the peptide can be purified using gel filtration to give the desired product.
  • the acetamidomethyl (Acm) thiol protecting group on the peptide may be removed using mercury (II) aceute with water/acetonitrile (1:1; 0.1% TFA) as solvent followed by precipitation of the mercury with 2-mercaptoethanol.
  • the resulting free thiol peptide can be purified using gel filtration to give the desired product.
  • NBC2 (thiol form) was synthesized by a standard solid state method and purified by hplc as described in Example 2.
  • the peptide was treated with a 50 molar excess of 2,2 ' -dithiopy ridy 1 disulphide and the S-pyridyl derivative purified by gel filtration. A 5x molar excess of this intermediate was then reacted with the reduced thiol of the deprotected fusogenic peptide and the conjugate isolated by ion-exchange chromatography.
  • the FPl was de ⁇ vauzed with a two molar excess of bromoacetyl-hydrazide which places a hydrazide residue on the C-terminal thiol. After purification by hplc this FPl intermediate was reacted with a 5 x molar excess of periodate oxidized NBC 12.
  • NBC 12 was oxidized by the method described in Example 7.2.1.1.
  • the resulting conjugate contains a hydrazone linkage between the FP and NBC2. Theoreucally this linkage is acid-labile, and will be broken under the acidic conditions in the endosome, releasing free FPl from the condensed DNA-conjugate complex.
  • the hydrazone group may be reduced with sodium borohydride to a stable hydrazide bond
  • This peptide was synthesized by standard solid phase automated methods as described in Example 8.1.1.
  • protease-cleavable sequence "GFLG" [Gly-Phe-Leu-Gly] present in the amino acid sequence of the peptide FPI3 (see above) was cleaved using the aspartic protease Cathepsin D. Othespsin D activity is found in the endosomal compartment. Complexes containing conjugated FPI3 peptide may have increased transfection efficiency compared to those containing conjugated FPI3, since in theory the FPI3 sequence would be cleaved in the endosome, thus allowing the peptide to fuse with the endosomal membrane.
  • the free peptide has been shown to have high fusogenic activity [Wagner et al [1992] Proc.Natl.Acad.Sci U.S.A. 89, 7934-38.
  • the peptide was cleaved rapidly at pH 5.0 and very slowly at pH 7.0, as monitored by gel filtration analysis of the cleaved peptide.
  • the peptide bond (*) between the phenylalanine and leucine of the "GFLG" sequence is hydrolysed by Cathepsin D to give two fragments of molecular weight 2370 and 622.
  • the larger N-terminal fragment has an absorbance at 280 nm but the smaller fragment does not.
  • the cleavage reaction can therefore be monitored by measuring the A 21t0 of the eluent obtained from high resolution gel filtration.
  • 450 ⁇ l of 1 mg/ml peptide solution in 0.15 M NaCl was prepared using either 50mM sodium citrate, pH 5.0; 50mM sodium curate, pH 6.0; or 50mM HEPES, pH 7.0.
  • Th i s pept i de may be conjugated to NBC pept i des by the TM ⁇ Budapest ⁇
  • the coniugate was purified on a SP-sepharose column.
  • the column was equilibrated in 25 M phosphate pH 7.4 and the cmde conjugate loaded. Unconjugated horseradish peroxidase washed straight through the column The conjugate was eluted with a salt gradient of 0-1.5 M.
  • the brown coloured fractions from the gradient were pooled and loaded onto a Sephacryl S-300 column equilibrated in 25 mM phosphate pH 7.4; 0.6 M NaCl Once again the brown coloured fractions eluting from the column were pooled.
  • the purified conjugate was too dilute for use straight from the column. Therefore it was dialysed against three changes of 5 L of distilled water and then freeze dried The resulting powder was dissolved in approximately 0.8 ml water and filtered to remove insoluble material.
  • the transfection assay described in Example 4 using Jurkat cells was employed with the following deviations from the gene ⁇ c method.
  • 50 ⁇ g DNA were condensed in 0.6M sodium chloride, 25mM sodium phosphate buffer, pH 7.4, 2% PEG 10000 with 2.5mg of conjugate. After incubation overnight at 4°C the complex solution was diluted 1 4 with a solution of 37mM sodium chloride, 25mM sodium phosphate buffer, pH 7.4. 10% PEG 10000 containing Lipl3 at a concentration of 0.15 ⁇ g ⁇ g DNA.
  • 2.5 ⁇ g DNA per assay point (2x 10 6 cells) were used. 41606
  • the cells were fixed in 0,5% glutraldhyde for 10 minutes at room temperature. The cells were then washed with phosphate buffered saline. The cells were then stained with Vectar Labs AEC (Vectar Labs Inc. Burlingaeme, CA. U.S.A.) for lOmin. The stained cells were examined under an optical microscope x20 magnification. The damples treated with the conjugate showed a delivery frequency of approximately 20% as evidenced by complete black staining of the cells involved. No blacj staining was observed in the control sample
  • NBC2 contains the SV40 T antigen nuclear localization signal. This sequence is known to promote the nuclear localization of proteins which contain the sequence (Roberts.B ( 1989; Biochim. Biophys. Acta 1008. 263). Peptide NBC5 has an identical stmcture to NBC2 except the sequence of the nuclear localization sequence is reversed. Reversal of the sequence motif is known to abolish nuclear localizauon properties.
  • the relative transfection potencies of NBC2 and NBC5 were compared in the standard non-targeting transfection assay (as described in Example 4) using Jurkat cells.
  • M9 includes the nuclear localization domain of hnRNP Al.
  • the sequence of M9 is as follows:
  • the sequence was modified by the addition of an N-terminal threonine residue and a C-terminal cysteine residue to permit chemical coupling to NBC2.
  • the peptide was synthesized using a Millipore 9050 plus peptide synthesizer in extended synthesis cycle mode ( 1 hour couplings). Fmoc-Cys(Acm)-O-PEG-PS- was used.
  • the acetamidomethyl (Acm) thiol protecting group on the peptide may be removed using mercury (II) acetate with water/acetonitrile (1:1; 0.1% TFA) as solvent followed by precipitation of the mercury with 2-mercaptoethanol.
  • the resulting free thiol peptide can be purified using gel filtration to give the desired product.
  • Coupling of the M9 sequence to NBC2 is best achieved by oxidation of the M9 sequence with periodate to form an N-terminal aldehyde functionality.
  • the thiol of NBC2 is deblocked and reacted with bromoacetylhydrazide.
  • the hydrazide of NBC2 is then reacted with oxidized M9 and the conjugate purified by hplc.
  • HIV is a retrovims which is able to infect non-dividing cells.
  • the HIV Matrix protein is one of the proteins responsible for this property and the protein has two nuclear localization sequences and is thought to be responsible for transport of the vims through the nuclear pore (GaJlay et al. (1995) Cell 80, 379; (1996) 83, 859).
  • the sequence motifs thought to be responsible for the effect have been coupled in the design of a synthetic peptide sequence called MAT1 which has the following stmcture-
  • the peptide was synthesised using a Biosearch 9050 plus Pepsynthesizer in extended synthesis cycle mode using Fmoc-Cys(Acm)-O-PEG-PS-Resm with DMF as solvent Deprotection of the N-terminal Fmoc -group before each coupling was achieved using a solution of 20% piperidine in DMFdmin at a high flow rate followed by 10 nun at 3ml/m ⁇ n) The ammo acids were coupled in four fold excess using 0-(lH- benzot ⁇ azo- 1 -yD-tetramethyluronium tetrafluoroborate (TBTU)/l-hydroxybenzot ⁇ azole and N-ethyldiisopropylamme as activating agents.
  • TBTU 0-(lH- benzot ⁇ azo- 1 -yD-tetramethyluronium tetrafluoroborate
  • the pepude was cleaved from the resin using a TFA/water/phenol/th ⁇ oan ⁇ sole/1,2- ethanedithiol (82.5: 5: 5: 5: 2.5) mixture ( 10ml of mixture for every gram of resin to be cleaved).
  • the resin was then removed by filtration and washed 3 times with TFA.
  • the combined filtrate and washings were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to give the cmde peptide.
  • the cmde peptide was dissolved in a small amount of 1% acetic acid in water and applied to a Sephadex G25 (superfine) column of an appropriate size arid eluted using the same 1 % Acetic acid solution
  • the fractions containing the peptide as shown by analyucal reverse phase hplc, were pooled and lyophilised. Further purification was achieved by preparative reverse phase hplc using a Dynamax 83-221-C column and an appropriate gradient of Aceton ⁇ t ⁇ le(0.1 % TF ⁇ ) in Water(0.1% TFA) over 20 mm.
  • the fractions containing the peptide were pooled, the acetonitrile evaporated in vacuo and lyophilised.
  • the peptide was then desalted by elution on a sephadex G25 superfine column ( 1.6x30cm) with 1% acetic acid in water. The resulting fractions were analysed by reverse phase hplc, pooled and lyophilised as above.
  • the acetamidomethyl protecting group on the cysteine resin may be removed by treatment with Mercu ⁇ c acetate in 30% aceuc acid water.
  • the product of this reaction may then be further purified by gel filtration on G25 as described above.
  • the free thiol group may the be used as a handle to attach the peptide via a linker to an NBC.
  • oxidation of the N-terminal threonine moiety with periodate, as described for NBC 12 furnishes the N-terminal glyoxal derivative which can be coupled to an appropriate peptide hydrazide
  • the homeodomain of the Antennapedia gene product has been shown to have nuclear localization properties (Derossi et al [ 1994] J. biol. Chem. 269, 10444).
  • the sequence of the domain thought to be responsible for this property has been identified and is inco ⁇ ortated in the following peptide
  • the peptide was synthesised using a Biosearch 9050 plus Pepsynthesizer in extended synthesis cycle mode using Fmoc-Cys(Trt)-O-PEG-PS-Resin with DMF as solvent. Deprotection of the N-terminaJ Fmoc-group before each coupling was achieved using a solution of 20% piperidine in DMF( 1 min at a high flow rate followed by 10 min at 3ml/min).
  • the amino acids were coupled in four fold excess using O-(lH- be ⁇ zotriazo-l-yl)-tetramethyluron ⁇ um tetrafluoroborate (TBTU)/l-hydroxybenzotriazole and N-ethyldiisopropylamine as activating agents.
  • the coupling times were set at lhour throughout the synthesis.
  • the peptide was cleaved from the resin using a TFA/water/phenol/thioanisole/1,2- ethanedithiol (82.5: 5: 5: 5: 2.5) mixture (lOmJ of mixture for every gram of resin to be cleaved).
  • the resin was then removed by filtration and washed 3 times with TFA.
  • the combined filtrate and shing were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to give the cmde peptide.
  • the cmde peptide was dissolved in a small amount of 25mM ammonium bicarbonate in water and applied to a Sephadex G25 (superfine) column of an appropriate size and eluted using the same 25mM ammonium bicarbonate in water.
  • the fractions containing the peptide as shown by analytical reverse phase hplc, were pooled and lyophilised.
  • the thiol group may be blocked activated at this stage with Dipyridyldisulphide if desired
  • the peptide was desalted by elution on a sephadex G25 superfine column (1.6x30cm) with 25mM ammonium bicarbonate in water The resulting fractions were analysed by reverse phase hplc, pooled and lyophilised as above.
  • Neutral hydrophillic polymers such as PEG are known to effect the in vivo stability of particles and proteins in the blood stream. Incorporation of such polymers would therefore be of advantage in the development of parenteral gene delivery systems.
  • the pegylated peptide is purified by cation exchange chromatography as described in Example 2, and incorporated into the synthetic vims like particles as described in Example 4. The amount of pegylated peptide required to increase the in vivn transfection will vary with the cell type of the target cell.
  • mPEG-MAL monomeihoxy-PEG S000
  • Sheatwater Polymers Inc. Huntsville. Al. U.S.A. and has been used to prepare a totally defined PEG-NBC9 conjugate
  • the single cysteine thiol of NBC 12 was deprotected and reacted with the rnaleimide function on the PEG as follows: mPEG- MAL was added at a 5-fold molar excess to 15 mg of the thiol form of NBC 12 in 25mM HEPES buffer, 0.15M sodium chloride (pH 7.4). The reaction was left at 25°C for 16h. 1606
  • the conjugate is "heavier" by Superdex Peptide analysis than NBC12. That the conjugate is covalently linked is supported by evidence from gel filtrauon with the Superdex Peptide column using 6M Guanidine.HCl as running buffer
  • Fmoc-NH-PEG(3400)-COOH A two fold excess of Fmoc-NH-PEG(3400)-COOH is coupled to the HMB-PS resin using DCC and DMAP and recirculating overnight The remaining am o acids (Fmoc- Gly-OH.
  • Fmoc-Ser (O(Ac,-mann))-OH and Fmoc-Tyr('Bu)- OH) are coupled in four-fold excess using the standard 1 hour coupling cycle with TBTU/N,N'-ethyldiisopropylam ⁇ ne activation and deprotection using 2% DBU in dimethylformamide containing 2% pipe ⁇ dme.
  • High Salt Formulation Two alternative types of formulation which do not require complicated preparation procedures and which are stable have been developed : these types of formulation are called High Salt Formulation and Isotonic Formulation.
  • the Isotonic formulations require dilution with a solution, "the Diluent" just before administration.
  • the Diluent One of the major disadvantages of other methods of non-viral gene therapy, such as those involving the use of cationic liposomes or naked DNA is that the final product is not easily filter sterilized. This results in a requirement for aseptic processing during manufacture: an extremely expensive operation.
  • the invention described here allows for easy filter sterilization by methods commonly used and validated in the pharmaceutical industry.
  • the High Salt Formulation is an in vivo formulation which involves direct injection of a hypertonic solution of the gene delivery vehicle. This formulation is therefore limited to direct injection of relatively small volumes ⁇ 5ml into either specific tissues such as tumour tissue or i.v.
  • the Isotonic Formulation is useful for injection of large volumes or in ex vivo therapies.
  • This formulation is particularly useful for the preparation of complexes which contain components which bind to filtration membranes. It has been shown that the presence of PEG 3000- 10000 prevents irreversible adso ⁇ tion of Lip 13 to the filtration membrane
  • the NBC2 solution (1 vol) was slowly added to the DNA solution (lvol) with agitation using a IKA-Schuttler MT4 machine [ 300 ⁇ m ] at a rate of 0.1 vol per minute. This operation was earned out at room temperature. After incubation for lh at room temperature the complex was stored overnight at 4°C
  • the NBC 13 and Lip 13 solution ( 1 vol ) was slowly added to the DNA solution ( I vol) with agitation using a IKA-Schutiler MT4 machine [ 300 ⁇ m ] at a rate of 0.1 vol per minute This operation was earned out at room temperature. After lh at room temperature the compex was filter ste ⁇ hzed
  • the gene delivery complex is stored as a high salt formulation and diluted just prior to use
  • the concentrate Prior to assay or injection the concentrate was diluted with the PEG solution 1 volume of gene complex concentrate : 4 volumes of PEG diluent. The diluted preparation was then used within 24h.
  • the concentrate was diluted with the PEG solution 1 volume of gene complex concentrate 20 volumes of PEG diluent The diluted preparation was then used within 24h
  • a series of formulations of pRSVLuc DNA were prepared and stored in vials at -20°C and 4°C. At va ⁇ ous time intervals the preparations were assayed for transfection efficiency using Jurkat cells as described in Example 4
  • F i gure 27 shows that in all excep t t wo ca s . s , ⁇ . f , ep i o cases t he formulations are stable .
  • Example 14 Delivery of Oligonucleotides ta Ccj
  • the invention encompasses the delivery of oligonucleotides to cells in an analogous manner to plasmid DNA.
  • Two 17 base oligonucleotides were synthesized:
  • oligonucleotide was synthesized on a phosphorothiolate backbone and was labeled with biotin in the 5' position
  • BC1-2S-B 10 is a sense sequence oligonucleotide
  • BC1-AS- B 10 is an amisense oligonucleotide
  • the latter oligonucleotide would be expected to interact with the natural human BCI-2 gene and by inhibiting BC1-2 gene expression induce apoptosis in tumorigenic cell lines such as Jurkat
  • oligonucleotides Delivery of oligonucleotides was demonstrated by formulating BC1-2S-B10 and BC1- AS-B 10 into a synthetic vims-like panicle, as taught herein, and visualizing delivery to Jurkat cells
  • the oligonucleotides were condensed with 2 ⁇ g NBCB/l ⁇ g oligonucleotide (lOO ⁇ g oligonucleotides/ml) as described in Example 4 with the following modifications.
  • the buffer was 25mM sodium phosphate pH 7 4. 0 6M with respect to sodium chloride. After 16 h at 4.0°C.
  • the complex was dined to 20 ⁇ g obgonucleotide/ml in 10% PEG 10,000, 37.5mM sodium chloride, 25mM sodium phosphate buffer pH 6.5 containing 120 ⁇ m chloroquine 0.15 ⁇ g Lip 13/ ⁇ g oligonucleotide DNA 1 x 10 6 Jurkat cells per point were transfected with 2.5 ⁇ g oligonucleotide DNA. The cells were incubated for 4 h as described in Example 4 prior to replacing the transfectioa medium with buffer. After 24 h the cells were washed with phosphate buffered saline aad flushed for 10 min in 0.05% glutaraldehyde at room temperature.
  • the cells were again washed with phosphate buffered saline and then permeabilized with 0.1% trium x 100 for 2 min at 4.0°C. After again washing with phosphate buffered saline the cells were treated with Vectorstain ABC according to the manufacturers instructions (Ve ⁇ or Labo ⁇ iories, Burlington, C.A.). This kit contains an avidin (which bonds strongly to biotin), horseradish peroxidase conjugate. After 60 mm at room temperature the cells were washed with phosphate buffered saline and stored with Vectorstain AEC. The presence of horseradish peroxidase in the cells is indicated by a black color. After further washes with water the cells were examined at x 20 magnification by optical microscopy. The control cells were negative. Cells treated with synthetic virus-like panicle containing oligonucleotides were positive (black) at 8 frequency of 20%.
  • Panicles of the invention are also capable of high level transfection of primary cells of the hematopoietic system (Figs 28 and 29) In both cases the cells were incubated using the standard assay conditions except that 100 ⁇ M chloroquine was included in the transfection medium.
  • Fig 28 shows that the ant ⁇ -CD33-NBClcomp!ex can effect gene transfer to Peripheral Blood Mononuclear Cells at similar efficiencies to cell lines
  • the compa ⁇ son shows the transfection efficiency of ant ⁇ -CD33-NBCl against the CD33+ cell line K562 and pe ⁇ pheraJ blood mononuclear cells prepared from pe ⁇ pheral blood b> standard Ficoll gradient centrifugation Given the error between assays (% SEM-30%). the activities can be considered equivalent
  • Fig 29 shows that DNA complexed with ant ⁇ -CD7-NBCl can effectively transfer genes to fresh pe ⁇ pheraJ blood mononuclear cells and p ⁇ mary cultures of EL-2 stimulated T-Cells as well as cell lines (Jurkat)
  • the compa ⁇ son shows the activity of ant ⁇ -CD7-NBC2 conjugates on the CD7* cell line Jurkat (de ⁇ ved from a T-celi line) and fresh pe ⁇ pheraJ blood monocyies and pe ⁇ pheraJ blood monocytes activated by culturing under standard conditions in the presence of H-2 for 7 days
  • An important object of the invention is ⁇ o transfer exogenous nucleic acid to mammalian cells in vivo In this exjmplc. in vivo transfection of tumor cells by a Synthetic Virus like Panicle of the invention is unequivocally demonstrated.
  • a u ⁇ ne carcinoma model was used to demonstrate the efficiency of the synthetic virus like particle in vivo
  • Three BDFI male mice (a strain developed by the Paterson Institute of Cancer Research. Manchester U.K. by crossing C57B 16 with DBA2 mice) were implanted with cells of carcinoma line T50/80, a murine mammary carcinoma cell line which arose spontaneously in BDFI mice (Paterson Institute; Dodd et aJ 1989 B ⁇ tish J. Cancer 60, 164). 8 weeks after implantation each of the mice earned a 6-9mm diameter rumour mass on the ⁇ ght flank.
  • peptide NBC2 in the same buffer was added dropwise to the DNA at a rate of 0.1 vol/min. The complex was incubated overnight at 4°C. Lip2 at a final concentration of 0.3 ⁇ g/ ⁇ g DNA was then added to the complex mixture and incubated at 37°C for 30 minutes.
  • mice bea ⁇ ng T50/80 tumors were anaesthetized with ether and the tumour mass injected with 20 ⁇ l of the following solutions: animal (a) HEPES buffer containing 7.14 ⁇ g plasmid DNA; animal (b) delivery complex containing 7.14 ⁇ g and animal (c) was injected with the same solution as animal (b) with an additional 0.24 ⁇ l of a lOmM solution of chloroquine dissolved in the formulation buffer.
  • mice were sac ⁇ ficed by cerebral dislocation 48h after injection. Tumours were removed by dissection snap frozen in liquid nitrogen and sectioned (14 ⁇ rn sections cut through the center of the tumour mass) before fixing in 0.25% glutaraldehyde in phosphate buffered saline Sections were stained for ⁇ -galactosidase activity for 24h in
  • Fig. 24 presents photographs taken of sections through tumour tissue transfected with plasmid DNA coding for the repo ⁇ er gene lacZ. which leads to the expression of the enzyme ⁇ - galactosidase.
  • the sections were prepared as described in the text and stained to show the location of cells expressing ⁇ -galactosidase.
  • Slides were examined by and recorded using a standard microscope fitted with bright field optics. Slide 1 shows the results obtained from the tumour earned by Animal A which was injected with naked plasmid DNA. Slide 2 shows the results of the experiment performed with Animal B. This animal was injected with the complex prepared as described in the text. Slide 3 was taken from the tumour carried by Animal C whose tumour was injected with the same 41606
  • mice Four BDFI male mice were implanted subcuianeously with cells of the T50/80 carcinoma line (Moore, Jpn J Cancer Res 79. 236-243 (1988). 8 weeks after implantation each of the mice earned a tumour mass of diameter 6-9mm on the ⁇ ght flank
  • An equal volume of peptide NBC 13 ai 400ug/mJ in the same buffer was added dropwise to the DNA at a rate of 0.1 vol /min
  • the complexes were incubated overnight at 4°C then equal volumes of each were mixed together.
  • the 50.50 mixture was then diluted to 5 ⁇ g DNA/mJ into RPMI- based medium containing 50 ⁇ g/ml human transfemn and lmg/ml human serum albumin (RAT medium), with or without 10% PEG 8000.
  • RAT medium 50 ⁇ g/ml human transfemn and lmg/ml human serum albumin
  • PEG 8000 human serum albumin
  • LIP 13 was then added to the complex mixture to a final concentration of O. ⁇ g LIP13 per ⁇ g DNA, and finally chloroquine was added to 120 ⁇ M
  • These dual vector formulations were then administered to animals by injecting with 20 ⁇ l complex, giving a total dose of 0.1 ⁇ g plasmid DNA per tumour.
  • Fig 31(a) shows that in Tumour 1 , injection of DNA-NBC13 complexes diluted into RAT resulted in poor transfection efficiencies with only the occasional cell or small group of cells staining blue in just a few of all sections taken.
  • Figure 31(c) shows that injection of DNA-NBC13 complexes diluted into [RAT+ 10% PEG 8000 + 0.6 ⁇ gLIP13/ ⁇ gD A] resulted in the efficient transfection of large groups of cells and these clusters were visible throughout the tumour mass, indicating that the complexes were well dispersed throughout the tumour upon injection. Many isolated single cells and clusters of cells were visible in sections taken at many different positions throughout the tumour, again indicating efficient dispersion of complexes upon injection and efficient gene transfer mediated by the delivery system
  • Figure 31 (d) shows that injection of DNA-NBC13 complexes diluted into [RAT- 1 - 0 6 ⁇ gLIP13/ ⁇ gDNA] results in transfection of small groups of cells, and is less efficient than when this formulation is supplemented with 10% PEG 8000
  • Figure 31(e) shows a section of the same tumour in Figure 4 in which very efficient delivery to a layer of cells close to the surface of the tumour was observed with DNA-NBC13 complexes diluted into [RAT+0.6 ⁇ gLIP13/ ⁇ gDNA] Only one such region was observed in this tumour, nevertheless the level of expression in this region was high
  • Figure 31(f) shows a section of uniniected T50/80 tumour which stained negative for lacZ expression
  • the blue cells are intermingled with unstained cells, but encompass an area of -20% of the field of view.
  • Approximately 30 foci of blue staining material, each focus representing a small cluster of cells or single cell can be seen in this figure.
  • An area of faint blue staining can also be seen in the middle of the top edge of the figure.
  • An area of faint blue staining can also be seen in the middle of the top edge of the figure, indicating a further patch of transfected cells.
  • the nucleic acid condensing peptides and the nucleic acid to be delivered to cells may be formulated separately for parenteral administration or as the synthetic virus like panicle In the latter case the synthetic virus like panicle may be assembled j ust prior to use
  • the nucleic acid includes a gene whose expression would have some beneficial therapeuuc effect on the cells of the recipient
  • the therapeutic nucleic acid, in condensed form be less than about lOOnm. i.e., in the size range of approximately 1-lOOnm, or less than approximately 50kb in length
  • the nucleic acid may be in the form of plasmid DNA, either linear or circular, or in the form of a DNA fragment
  • therapeutic genes are well known in the an and include but are not iimited to the ⁇ -glucocerebrosidase gene, the Bruton's thymidme kmase gene, genes encoding cvtokines. such a TNF. interleukins 1 - 12, mterferons ( ⁇ , ⁇ , ⁇ ) , F, receptor. and T-cell receptor
  • the DNA also include marker genes, such as drug resistance genes, the ⁇ -galactosidase gene, the dihydrofolate reductase gene, and the chloramphenicol acetyl transferase gene
  • the pepudes and DNA are exchanged into isotonic phosphate free buffer and sterile filtered through a 045 or 0 22 ⁇ filter
  • the formulated solution or synthetic virus like panicle (a mixture of the pepude coniu ⁇ rated to a selected funcuonaJ group. DNA and free condensing pepude ) mav be ste ⁇ lc filled and aliquotted into suitable vials
  • the vials may be stored at 4°C. 20°C or 80°C or alternatively the DNA, peptide or synthetic virus like panicle may be freeze dried from a buffer containing an appropriated earner and bulking agent. In these cases, the dosage form is reconstituted with a ste ⁇ ie solution before administration
  • nucleic acid containing a gene of physiological impo ⁇ ance such as replacement of a defecuve gene or an additional potentially beneficial gene function, is expected to confer long term genetic modification of the cells and be effecuve in the treatment of disease
  • a patient that is subject to a viral or genetic disease may be treated in accordance with the invention via in vivo or ex vivo methods.
  • a delivery vehicle of the invenuon can be administered to the patient, preferably in a biologically compatible soluuon or a pharmaceutically acceptable carrier, by ingestion, in j ecuon. inhalation or any number of other methods.
  • a composition including a synthetic virus like panicle will be administered in a single dose in the range of 10 ng - 100 up kg body weight, preferably in the range of 100 ng - l ⁇ ug/kg body weight, such that at least one copy of the therapeutic gene is delivered to each target cell.
  • the therapeutic gene will, of course, be associated with approp ⁇ ate regulatory sequences for expression of the gene in the target cell.
  • Ex vivo treatment is also contemplated within the present invention
  • Cell populations can be removed from the patient or otherwise provided, transduced with a therapeutic gene in accordance with the invention, then re troduced into the patient.
  • ex vivo cell dosages will be determined according to the desired therapeutic effect balanced against any deleterious side -effects. Such dosages will usually be in the range of 10"- 10" cells per patient, daily weekly, or intermittently; preferably 10 6 - 10 7 cells per patient
  • a synthetic virus-like panicle according to the invention may be used to treat X-lmked ⁇ -globulmemiu
  • the condensed nucleic acid in the virus-like parucle will contain the Bruton s tyrosine kmase gene (Vetne et al., 1993. Nature 361 .226-233). which is earned on a 2 1 kb fragment delineated by the Pvul site at position (+33) and the Hindlll site at position 1+2126).
  • the plasmid also may include sequences which confer position independent, tissue specific gene expression, as taught in PCT/GB88/006 5
  • the therapeutic gene may also encode a splice site and pol> A tail, which may include po ⁇ ions of the human ⁇ globm locus splice and poly A signals: i.e.. a BamHI Xbal 2.8 kb ⁇ sphcc/pol A flanking sequence containing exon 2 IVSII - exon 3 - - polyA sequences
  • a synthetic vims-like panicle containing the Bruton's tyrosine k ase gene is assembled as described herein and used to treat X-hnked ⁇ -globulinemia by introducing the construct directly into a patient for in vivo gene therapy or into pre-B cells for ex vivo therapy, as described in Ma ⁇ ensson et al.: Eur. Jour. Immunol. 1987, 17: 1499; Okabe et al., Eur. Jour. Immunol. 1992. 22:37; and Banerji et al., Cell 33:729, 1983. and administering the transfected pre-B cells into a patient afflicted with X-linked ⁇ - globulinemia.
  • a synthetic virus-like panicle for treatment of X- linked ⁇ -globuline ⁇ ua will include a Iigand for targeting of a preB cell.
  • Iigand for targeting of a preB cell.
  • Such ligands are well-known in the an and will be specific for and capable of targeting one or more of 1606
  • CD9 CD10
  • CD19 CD20
  • CD22 CD24
  • CD38 CD40.
  • CD72. and CD74 CD74.
  • a synthetic virus-like panicle described herein also may be used for treatment of Gaucher's disease.
  • Gaucher's disease stems from one of two different geneuc mutations.
  • Gaucher's type 1 is a CGG --> CAG mutation, which results in an Arg — >
  • Gaucher lypc 2 is j CTG -> CCG mutation, which results in a Leu --> Pro substitution at position 444 of the Z-glucocerebrosidase polypeptide (Tsuji, NEJM 316:570, 1987)
  • the presence of a : ⁇ -glucocerebrosidase gene encoding a wild type polypeptide is believed to substantially conect Gaucher's disease.
  • a therapeutic nucleic acid useful according to the invention includes the ⁇ -glucocerebrosidase gene, as described in Horowitz et al., 1989, Genomics 4:87-96, which is earned, as disclosed in Horowitz et al., on a 9722 base pair fragment extending from a BamHI sue in exon I to an EcoRV site 31 to polyadenylauon site This fragment contains 1 1 exons and all intervening sequences, with translational sta ⁇ in exon 2 Sequences conferring position-independent and ussue-specific gene expression may be included in the construct and are earned on an 1 1.8 kb Xhol - Sad fragment from plll.lyx construct described in Bonifer et al., 1990, Euro. Mol Biol Org Jour 9.2843
  • a syntheuc virus-like panicle containing the ⁇ -glucocerebrosidase gene is assembled as described herein and used to treat Gaucher ' s disease by introducing the virus-like panicle direct!) into the host for in vivo treatment, or into isolated macrophages for ex vivo . s described in Immunology and Cell Biolog) . 1993. Vol 71. pages 75-78 and introducing the transfected macrophages into a patient afflicted with Gaucher ' s disease Expression of the wild type transgene in a patient afflicted with Gaucher's disease should result in c ⁇ nection of the diseased state.
  • the synthetic virus-like panicle will contain a Iigand that specifically targets a cell surface antigen on a macrophage
  • Iigand that specifically targets a cell surface antigen on a macrophage
  • Such ligands are well-known in the an, for example, monoclonal antibody having specificity for and capable of targeting one or more of the following cell surface markers CD14, CD16, CD26, CD31, CDw32, CD36, CD45RO, CD45RB.
  • the cells targeted for in vivo or ex vivo gene transfer in accordance with the invention include any cells to which the delivery of the therapeutic gene is desired. Such cells will bear a cell surface marker for which a corresponding specific Iigand is available or can be prepared to allow for cell-specific targeting according to the invention.
  • cells of the immune system such as T-cells, B-cells, and macrophages, hematopoietic cells, and dendritic cells, each cell of which bears one or mo. wll-kncn cd, srfa, ua ⁇ ea havlng ⁇
  • the following example describes transfection of mammalian cells using a synthetic virus like particle in which a lipidated nucleic acid condensing peptide is present, which particle was prepared according to a novel procedure which includes preincubation of the synthetic virus like particle with chloroquine.
  • the synthetic virus like particle allows nucleic acid delivery to be optimized further by increasing binding of chloroquine through elevation of the level of lipophilic substituents in the synthetic virus like particle.
  • the range of preincubation concentrations useful according to the invention are generally from 10 ⁇ M to 70mM
  • the maximum amount of chloroquine administered with the synthetic virus like particle in vivo should not exceed 3.5 mg kg body weight.
  • the final concentration of chloroquine after dilution from the formulation should not exceed 200 ⁇ M.
  • Transfection efficiency in vitro can also be increased by extending the time period to which the target cells are exposed to the synthetic virus like particle in the presence of chloroquine.
  • Figure 23 shows the effect of increasing the incubation time on transfection of Jurkat cells by Lip2 synthetic virus like particles in the presence of 120 ⁇ M chloroquine.
  • solutions of NBC 1 were either made up at a concentration of 200 ⁇ g/ml in 25 mM Hepes, 0.85 M sodium chloride, pH 7 4 containing 0.7 M sodium chloride or in the same buffer containing 5% PEG (Sigma Ltd., Poole, Dorset). Each solution was mixed with an appropriate amount of 3.5 mg/ml RSVLUC plasmid DNA to give a final concentration of 100 ⁇ g/ml plasmid DNA and the mixture incubated at room temperature for 60 min.
  • the synthetic virus like particles were then diluted either directly into RPMI medium containing 1 x 10* Jurkat cells and 120 ⁇ M chloroquine (25 ⁇ l DNA synthetic virus like particle into 1ml of cell culture medium) or into 25 mM Hepes, 0.85 M sodium chloride, pH 7.4 containing 10% PEG to a final concentration of 25 ⁇ g DNA/ml. After 30min at room temperature, the solutions in Hepes buffer were each diluted into RPMI containing 1 x 10 6 Jurkat cells and 120 ⁇ M chloroquine ( 100 ⁇ l DNA synthetic virus like particle into 1 ml of cell culture medium).
  • the cells were centrifuged, the medium removed and the cells resuspended in 2.5 ml of RPMI medium containing 10% fetal calf serum. After 24h, the cells were collected, washed, and lysed and the level of luciferase expression determined as described above

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Abstract

Cette invention est fondée sur la découverte d'une particule de synthèse du type virus contenant une pluralité de peptides capables de condenser de l'acide nucléique et de l'acide nucléique condensé. La pluralité de peptides présente un faible indice de polydispersion et chaque peptide est un hétéropeptide. L'acide nucléique peut coder des séquences possédant un intérêt thérapeutique. La particule de synthèse du type virus s'assemble elle-mème et peut être conçue de manière à pouvoir cibler une cellule ou un type de tissu particulier et apporter l'acide nucléique devant être incorporé dans les séquences chromosomiques ou extrachromosomiques des cellules ou des tissus ciblés.
EP96917590A 1995-06-08 1996-06-10 Compositions pharmaceutiques ameliorees utilisees pour la therapie genique Withdrawn EP0831922A2 (fr)

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US12495P 1995-06-08 1995-06-08
US124 1995-06-08
GBGB9513399.7A GB9513399D0 (en) 1995-06-30 1995-06-30 Improved pharmaceutical compositions for gene therapy
GB9513399 1995-06-30
GBGB9519304.1A GB9519304D0 (en) 1995-09-21 1995-09-21 Improved pharmaceutical compositions for gene therapy
GB9519304 1995-09-21
US428595P 1995-09-25 1995-09-25
US4285 1995-09-25
US895295P 1995-12-19 1995-12-19
GB9525955 1995-12-19
US600895 1995-12-19
GBGB9525955.2A GB9525955D0 (en) 1995-12-19 1995-12-19 Improved pharmaceutical compositions
US1153196P 1996-02-12 1996-02-12
US11531 1996-02-12
US66023196A 1996-06-07 1996-06-07
US660231 1996-06-07
PCT/GB1996/001396 WO1996041606A2 (fr) 1995-06-08 1996-06-10 Compositions pharmaceutiques ameliorees utilisees pour la therapie genique

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EP1054694A2 (fr) 1998-02-13 2000-11-29 Selective Genetics, Inc. Procedes de melange de flux en parallele, appareils de preparation de vecteurs pour la therapie genique et compositions preparees de cette maniere
WO1999042091A2 (fr) 1998-02-19 1999-08-26 Massachusetts Institute Of Technology Compositions d'apport dans des cellules
AU771111B2 (en) 1998-07-21 2004-03-11 Emd Millipore Corporation A polynucleotide comprising a ubiquitous chromatin opening element (UCOE)
US6927278B1 (en) 1998-09-01 2005-08-09 Trustees Of The University Of Pennsylvania Peptide scaffolds for transfer of molecules into eukaryotic cells
WO2000012114A1 (fr) * 1998-09-01 2000-03-09 The Trustees Of The University Of Pennsylvania Structures peptidiques permettant de transferer des molecules dans des cellules eukariotes
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