EP1285081A2 - Procede ameliore de transfection - Google Patents

Procede ameliore de transfection

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Publication number
EP1285081A2
EP1285081A2 EP01931977A EP01931977A EP1285081A2 EP 1285081 A2 EP1285081 A2 EP 1285081A2 EP 01931977 A EP01931977 A EP 01931977A EP 01931977 A EP01931977 A EP 01931977A EP 1285081 A2 EP1285081 A2 EP 1285081A2
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EP
European Patent Office
Prior art keywords
cells
cell
transfection
nucleic acid
vector
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Withdrawn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
EP01931977A
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German (de)
English (en)
Inventor
Stephen L. Institute of Child Health HART
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ICH Productions Ltd
Original Assignee
ICH Productions Ltd
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Filing date
Publication date
Priority claimed from GB0013090A external-priority patent/GB0013090D0/en
Priority claimed from GB0013089A external-priority patent/GB0013089D0/en
Application filed by ICH Productions Ltd filed Critical ICH Productions Ltd
Publication of EP1285081A2 publication Critical patent/EP1285081A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the present invention relates to an improved method of transfecting cells.
  • transfection is used herein to denote the introduction of a nucleic acid into a cell.
  • the nucleic acid may be of any origin, and the recipient cell may be prokaryotic or eukaryotic.
  • Gene therapy and gene vaccination are techniques that offer interesting possibilities for the treatment and/or prophylaxis of a variety of conditions, as does anti-sense therapy. Such techniques require the introduction of a DNA of interest into target cells. The ability to transfer sufficient DNA to specific target cells remains one of the main limitations to the development of gene therapy, anti- sense therapy and gene vaccination. Both viral and non-viral DNA delivery systems have been proposed. In some cases RNA is used instead of DNA.
  • Receptor-mediated gene delivery is a> non-viral method of gene transfer that exploits the physiological cellular process, receptor-mediated endocytosis to internalise DNA.
  • Examples include vectors targeted against insulin receptors, see for example, Rosenkranz et al Experimental Cell Research 199, 323-329 (1992) , asialoglycoprotein receptors, see for example, Wu & Wu, Journal of Biological Chemistry 262, 4429- 4432 (1987) , Chowdhury et al Journal of Biological Chemistry 268, 11265-11271 (1993), and transferrin receptors, see for example, Ciriel et al, Proc. Natl. Acad. Sci. USA 88, 8850- 8854 (1991) .
  • vectors include monoclonal antibodies targeting receptors on neuroblastoma cells (Yano et al, 2000), folate conjugated to liposomes (Reddy & Low 2000, Reddy et al . 1999), galactose for targeting liver cells (Han et al . 1999 Bettinger et al . 1999) and asialogylcoprotein, also for liver cells (Wu et al . 1991).
  • Receptor-mediated non-viral vectors have several advantages over viral vectors. In particular, they lack pathogenicity; they allow targeted gene delivery to specific cell types and they are not restricted in the size of nucleic acid molecules that can be packaged. Gene expression is achieved only if the nucleic acid component of the complex is released intact from the endosome to the cytoplasm and then crosses the nuclear membrane to access the nuclear transcription machinery. However, transfection efficiency is generally poor relative to viral vectors owing to endosomal degradation of the nucleic acid component, failure of the nucleic acid to enter the nucleus and the exclusion of aggregates larger than about 150nm from clathrin coated vesicles.
  • Integrins are a super-family of heterodimeric membrane proteins consisting of several different ⁇ and ⁇ subunits. They are important for attachment of cells to the extracellular matrix, cell-cell interactions and signal transduction. Integrin-mediated cell entry is exploited for cell attachment and entry by a number of intracellular pathogens including Typanosoma cruzi (Fernandez et al . , 1993), adenovirus (Wickham et al . , 1993), echovirus (Bergelson et al . , 1992) and foot-and-mouth disease virus
  • Targeting of non-viral vectors to integrins therefore, has the potential to transfect cells in a process that mimics infection of cells by pathogens and avoids the size limitation imposed by clathrin-coated vesicles in receptor-mediated endocytosis.
  • a further advantage of integrin-mediated vectors is that a large number of peptide ligands for integrin receptors have been described, including sequences derived from natural protein ligands [Verfaille, 1994; Wang, 1995; Wunschz, 1991; Pierschbacher, 1984; Massia, 1992, Clements et al . 1994 & Lu et al, 1993] or selected from phage display libraries (Koivunen et al . 1995; 1993; 1994; O'Neil et al . 1992; Healy et al 1995; Pasqualani et al . 1995).
  • the conserved amino acid sequence arginine-glycine-aspartic acid is an evolutionarily conserved feature of many, but not all, natural integrin-binding ligands such as extracellular matrix proteins and viral capsids. Peptides, particularly those containing cyclic-RGD domains can also bind integrins. Peptides containing cyclic-RGD domains are particularly suitable ligands for vectors since they bind to integrins with higher affinities than linear peptides (Koivunen et al . 1995). Hart et al .
  • the cyclic RGD-containing peptide GGCRGDMFGCGG [K] i6 [SEQ. ID.NO. :1] was synthesised with a sixteen-lysine tail for complex formation with plasmid DNA (Hart et al . , 1995) .
  • Significant levels of integrin-mediated gene expression were achieved in epithelial cell lines with the vector GGCRGDMFGCG [K] le [SEQ . ID .NO. : 2] (Hart et al . , 1995) and the vectors GGCRGDMFGC [K] X6 [SEQ. ID.NO. : 3] (W096/15811) .
  • ID.NO. : 4] which has the sixteen-lysine domain at the N-terminus and which is easier to syn-thesise than the prototype peptide (W096/15811 and Hart et al . , 1997) generated better transfection levels. Integrin mediated gene expression was generally achieved at levels of about 1 to 10%. The presence of chloroquine in the transfection medium gave some enhancement of transfection in some but not all cell lines tested.
  • WO 98/54347 discloses a mixture comprising an integrin- binding component, a polycationic nucleic acid-binding component, and a lipid component, and also discloses a complex comprising
  • nucleic acid especially a nucleic acid encoding a sequence of interest, (ii) an integrin-binding component,
  • the complex is a transfection vector.
  • integrated targeting vector integrin targeting complex
  • integrated targeting vector complex are used interchangeably hereafter to denote the complex described in W098/53437 and herein.
  • transfection efficiency did indeed drop dramatically, for example, from about 40 to 50% for sub-confluent cultures to about 5% to 8% for confluent cultures.
  • EGTA ethylene glycol-bis ( ⁇ -aminoethyl ether) -N,N,N' ,N' - tetraacetic acid
  • a calcium chalator Schott-Setrachloride
  • the proposed mechanism is different for the two viruses, but the role proposed for EGTA is the same: the exposure of viral receptors on the basolateral membranes of the epithelial cells.
  • the present invention is based on the surprising observation that the use of EGTA with a transfection vector comprising DNA, an integrin-binding component, a polycationic nucleic acid-binding component, and a lipid component, enhances transfection efficiency of confluent cells, which are slowly dividing or non-dividing i.e. substantially non-mitotic cells, in vi tro and in vivo.
  • transfection efficiency of airway epithelial cells in vi tro using two different reporter genes was increased about four-fold, and in vivo transfection of mouse lungs was also increased about four-fold.
  • the present invention provides a method of transfecting cells, especially confluent cells or other slowly dividing cells or non-dividing cells that are in contact with each other, that is to say, cells that are substantially non- mitotic, with a nucleic acid, which method comprises treating the cells with a receptor targeted vector comprising the nucleic acid and with an agent that disrupts cell-cell junctions .
  • the confluent cells or other slowly dividing cells or non- dividing cells that are in contact with each other may be in the form of a culture, for example, a confluent culture in vi tro. Methods for producing such cultures are well known, and any cells that can be grown to confluence in vi tro may be transfected according to the present invention.
  • confluent cells or other slowly dividing cells or non-dividing cells that are in contact with each other may be transfected in vivo.
  • a particular example of a tissue that comprises confluent, differentiated and hence substantially non-dividing cells and that has proved recalcitrant to transfection is the airway epithelium, a target for gene therapy for cystic fibrosis and asthma.
  • Other epithelial and endothelial tissues are also particularly suitable targets for transfection according to the present invention.
  • the cell-cell junctions that are to be disrupted in the method of the present invention are junctions between adjacent cells.
  • the exact types of junction may vary from tissue to tissue to cell type to cell type, and the junctions include gap junctions, tight junctions and adherence junctions.
  • the tight junction found in epithelia, is also known as zonula occuldens.
  • Tight junctions located at the apico-lateral border of the epithelial cells, produce the major permeability barrier regulating diffusion of solutes across the epithelium. Tight junctions are calcium-dependent and are formed by a transmembrane protein, occludin, see Anderson (1995) and Mitric (1998) .
  • Adherence junctions located just below the tight junctions, are formed by E- cadherin, a calcium-dependent adhesion molecule essential for the maintenance of cell-cell adhesion, see Takeichi (1990) , Takeichi (1991) .
  • the cell-cell junctions to be disrupted according to the present invention are not limited to junctions in any specific tissue or between any particular types of cells. Examples of junctions that may be disrupted include those found in certain types of tissues in vivo, for example, epithelial and endothelial tissues.
  • Agents that are capable of -disrupting cell-cell junctions are known, for example, calcium-chelating agents and calcium-binding agents, for example, EDTA (ethylenediaminetetra-actetic acid) or, especially, EGTA.
  • EDTA ethylenediaminetetra-actetic acid
  • EGTA is generally preferred as it is more specific for calcium than is EDTA. It is also better tolerated by cells, both in vi tro and in vivo .
  • a further approach is the use of antibodies to substances involved in cell-cell adhesion, for example, at gap junctions, tight junctions, or adherence junctions, for example, cadherins .
  • Antibodies, especially monoclonal antibodies, to such substances, for example, anti-cadherins, may be used as an agent capable of disrupting cell-cell junctions .
  • the cells are treated with the vector of choice in the usual manner for transfection using that vector.
  • the agent that disrupts cell-cell junctions may be used before the cells are treated with the vector but it is preferably to treat the cells with the agent and the vector at the same time or substantially the same time.
  • the agent that disrupts cell-cell junctions is used in amount effective to disrupt the junctions.
  • the concentration of EGTA is about ImM or less, for example, from about 0.5 mM to 1 mM. Higher concentrations may be used, but care should be taken with regard to toxicity. A concentration of about 1 mM is generally preferred for use in vi tro .
  • the concentration of EGTA may be about 25 mM to 200 mM, for example, 100 mM. Concentrations greater than 200 mM may be used, but again care must be taken with regard to toxicity at high concentrations, for example, 400 mM may be lethal. Concentrations of about 100 mM are generally preferred.
  • the cells may be transfected in vi tro or in vivo .
  • Transfection in vi tro is particularly useful for transfecting non-dividing cells with genes or anti-sense DNA of interest.
  • Such cells may be harvested for use, for example, for administration to a patient or for use for protein production.
  • such cells may be used in their confluent state, for example, in si tu, as disease models for drug testing. It is considered that confluent cells, which are generally non-dividing and may be differentiated, are often better models of tissues that are sub-confluent cells.
  • Cells may be grown to confluence in microwells, or using more sophisticated systems, for example, at an air-liquid interface.
  • Various systems for growing confluent cells for use as models for drug testing are being developed.
  • the ability to transfect cells in such systems is a great advantage .
  • Confluent cells and other slowly dividing or non-dividing cells that are in contact with each other, including differentiated cells, may be transfected in vivo .
  • the present invention provides such a method of transfection and also provides the use of an agent that disrupts cell-cell junctions and a receptor targeted vector in the manufacture of a medicament for the transfection of cells, especially confluent, or other slowly dividing or non-dividing cells that are in contact with each other, for example, substantially non-mitotic cells.
  • Such cells include, for example, endothelial or epithelial cells, for example, cells of the any part of the airway epithelium, including bronchial and lung epithelium, and the corneal endothelium.
  • the airway epithelium is an important target for gene therapy for cystic fibrosis and asthma.
  • the vector that may be used in the present invention is in particular any non-viral vector that is targeted to a cell- surface receptor.
  • many such vectors have been proposed, including vectors targeted to insulin, asialoglycoprotein and transferrin receptors, see above.
  • Further examples of vectors include monoclonal antibodies targeting receptors on neuroblastoma cells (Yano et al, 2000), folate conjugated to liposomes (Reddy & Low 2000, Reddy et al . 1999), galactose for targeting liver cells (Han et al . 1999 Bettinger et al . 1999) and asialogylcoprotein, also for liver cells (Wu et al . 1991) . It may be advantageous to use a vector that comprises a polycationic component.
  • Vectors targeted against integrin receptors have particular advantages, especially the vectors described in W096/15811 and, in particular, the transfection vector complexes described in W098/54347 and herein.
  • the nucleic acid may be obtained from natural sources, or may be produced recombinantly or by chemical synthesis. It may be modified, for example, to comprise a molecule having a specific function, for example, a nuclear targeting molecule.
  • the nucleic acid may be DNA or RNA. DNA may be single stranded or double stranded.
  • the nucleic acid may be suitable for use in gene therapy, in gene vaccination or in an anti-sense therapy.
  • the nucleic acid may be or may relate to a gene that is the target for particular gene therapy that is to say, a gene for newer treatment by gene therapy is desired, fore example, a gene having a mutation, or another defect, or a gene that is deficient, or a gene that is deficient, or a gene that is absent or is present in insufficient amounts or that is present in excess, any of which effects causes a disease or disorder.
  • the nucleic acid may be a molecule that can function as a gene vaccine or as an anti-sense therapeutic agent.
  • the nucleic acid may be or correspond to a complete coding sequence or may be part of a coding sequence or may be a control or regulatory element or may be or correspond to a genetic sequence comprising all or some elements selected from the coding sequence of a gene and the upstream and downstream non translated sequences, including control and regulatory elements.
  • the nucleic acid may encode a protein that is commercially useful, for example industrially or scientifically useful, for example an enzyme; pharmaceutically useful, for example, a protein that can be used therapeutically or prophylactically as a medicament or vaccine; or diagnostically useful, for example, an antigen for use in an ELISA.
  • Host cells capable of producing commercially useful proteins are sometimes called "cell factories" .
  • nucleic acid is a protein coding sequence.
  • the nucleic acid component is generally presented in the form of a nucleic acid insert in a plasmid or vector. In some cases, however, it is not necessary to incorporate the nucleic acid component in a vector in order to achieve expression. For example, gene vaccination and anti-sense therapy can be achieved using a naked nucleic acid.
  • the nucleic acid is generally DNA but RNA may be used in some cases, for example, in cancer vaccination.
  • the nucleic acid component may be referred to below as the plasmid component or component "D" .
  • the receptor targeted vector is especially an integrin targeted complex as described in W098/54347, that is to say, a complex comprising
  • nucleic acid especially a nucleic acid encoding a sequence of interest
  • the integrin-binding component is any component that is capable of binding specifically to integrins found on the surface of cells.
  • the integrin- binding component may be a naturally occurring integrin- binding ligand, for example, an extracellular matrix protein, a viral capsid protein, the bacterial protein invasin, a snake venom disintegrin protein, or an integrin-binding fragment of any such protein.
  • integrin-binding proteins and fragments thereof may be obtained from natural sources or by recombinant techniques, but they are difficult to synthesise and purify in large amounts, they require conjugation directly to DNA or RNA or to polycationic elements for DNA or RNA binding, and are immunogenic in vivo.
  • integrin-binding peptides it is preferable to use integrin-binding peptides, in particular because of their ease of synthesis, purification and storage, their potential for chemical modification, and their potentially low immunogenicity in vivo .
  • Examples of integrin-binding peptides are given in Verfaille, 1994 #635; Wang, 1995 #645; Wunschz, 1991 #539; Pierschbacher, 1984 #314; Massia, 1992 #86, Clements et al . 1994 & Lu et al , 1993; and in Koivunen et al . 1995; 1993; 1994; O'Neil et al . 1992; Healy et al 1995; and Pasqualani et al . 1995.
  • peptides containing the conserved amino acid sequence arginine-glycine-aspartic acid bind with high affinity to integrins. Accordingly, peptides comprising the RGD sequence are particularly useful .
  • the affinity between integrin and peptide ligands is influenced by the amino acid sequence flanking the RGD domain. In peptides having a cyclic region encompassing all or part of the region comprising the RGD sequence, the conformational freedom of the RGD sequence is restricted.
  • Such peptides generally have a higher affinity for integrin receptors than do their linear counterparts. Such cyclic peptides are particularly preferred.
  • Cyclic peptides may be formed by the provision of two cysteine residues flanking the RGD sequence in the peptide, thus enabling the formation of a disulphide bond.
  • a cysteine residue may be separated from the RGD sequence by one or more residues, for example, up to six residues, or may be immediately adjacent to the RGD sequence, although preferably both cysteines are not immediately adjacent to the ends of the RGD sequence .
  • Two further cysteine residues may be present, enabling formation of two disulphide bonds.
  • a peptide that consists of or comprises the sequence CRGDMFGC may advantageously be used as an integrin- binding peptide according to the present invention.
  • Examples of peptides that comprises the sequence CRGDMFGC and that are effective integrin-binding ligands are the peptides
  • the peptide GACDCRGDCFCA [SEQ. ID.NO. : 10] has the potential to form two disulphide bonds for stabilising the RGD loop. That peptide and others having the potential to form two RGD- stabilising disulphide bonds by the presence of two or more cysteine residues, may be particularly useful as integrin- binding ligands according to the present invention.
  • integrin-binding peptides contain the conserved RGD sequence.
  • the peptides GACRRETAWACA [SEQ. ID.NO. : 11] and GACRRETAWACG [SEQ. ID.NO. : 12] are integrin-specific peptides.
  • Other peptides comprising the sequence CRRETAWAC [SEQ. ID. O..-13] may be used, as may other non-RGD peptides, particularly those that have the potential for disulphide bond formation by the provision of two or more cysteine residues.
  • Peptide sequences may be designed on the basis of known ligands, for example, on the basis of integrin-binding domains of naturally-occurring integrin-binding ligands, or on the basis of known peptides that bind to integrins.
  • integrins are a family of heterodimeric pro- teins found on the surface of cells. They consist of several different and ⁇ subunits. Some integrins are found on may types of cells, others are more specific, for example, 5 and ⁇ v integrins are widespread and are found on a diverse range of cells. Integrin-binding ligands can vary in their affinity for different integrins. For example, GACRGDMFGCA [SEQ. ID. NO. : 9] (peptide 1) has affinity for ⁇ 5 and ⁇ v integrins but is non-specific (O'Neil et al . 1992, Hart et al. 1997). GACDCRGDCFCA [SEQ. ID.NO.
  • Peptide 7 ( ⁇ 4 ⁇ l) GAGPEILDVPST SEQ. ID.NO. : 16
  • Peptide 8 ( ⁇ 4 ⁇ l) GACQIDSPCA SEQ. ID. O. : 17
  • the inital two residues "GG” or "GC”, where present, are spacers.
  • a preferred embodiment of the present invention is based on the observation that a modified spacer has improved transfection efficiency.
  • the receptor-targeted vector is preferably a complex that comprises
  • nucleic acid especially a nucleic acid encoding a sequence of interest, (ii) an integrin-binding component,
  • a plycationic nucleic acid-binding component (iii) a plycationic nucleic acid-binding component, and (iv) a lipid component, in which complex the integrin binding component comprises an integrin-binding element and a spacer element, the spacer element being longer and/or more hydrophobic than the dipeptide spacers GG (glycine-glycine) and GA (glycine-alanine) .
  • GG glycine-glycine
  • GA glycine-alanine
  • Such a preferred spacer element is generally a peptide, that is to say, it comprises amino acid residues.
  • the amino acids may be naturally occurring or non-naturally occurring. They may have L- or D-configuration.
  • a preferred spacer element may be longer than a dipeptide. It may, for example, comprise three or more amino acids, for example, four or more, for example, five or more, for example, up to ten amino acids or more.
  • the amino acids may be the same or different, but the use of multiple lysine residues should be avoided in the preferred spacer as oligolysine sequences are the preferred polycationic nucleic acid-binding component of a complex of the present invention.
  • the preferred spacer may be more hydrophobic than the dipeptides GG and GA.
  • amino acids that are more hydrophobic than glycine and alanine may be used.
  • hydrophobic amino acids are well known and include ⁇ -amino hexanoic acid.
  • a preferred spacer may be either longer or more hydrophobic than the dipeptides GG and GA, or it may be both longer and more hydrophobic .
  • a spacer is generally at the N-terminus of the integrin-binding peptide.
  • the "integrin binding peptides" described above comprise both an integrin- binding peptide element and a spacer dipeptide GG or GC, see for example, the peptides of GGCRGDMFGC SEQ. ID.NO: 6, which comprises the integrin-binding element CRGDMFGC [SEQ. ID.NO. : 5] and the dipeptide spacer GG.
  • the preferred spacer element of the present invention takes the place of a GG or GA spacer in peptides described above.
  • integrin binding peptides include the following: CRGDMFGC [SEQ . ID .NO . : 27] ; CRGDMFGCG [SEQ. ID.NO. : 5] ; CRGDMFGCA [SEQ . ID .NO . : 28] ; CDCRGDCFCA [SEQ . ID .NO . : 29] ; CRRETAWACA [SEQ . ID .NO . : 30] ; CRRETAWAC [SEQ. ID.NO. : 13] ; CRRETTAWAC [SEQ . ID .NO . : 31] ; CRRETAWACG [SEQ . ID .NO.
  • a preferred spacer may be linked to any of the above peptides, preferably at the or terminus thereof.
  • the terms "integrin-binding component” and “integrin-binding peptide” and any other such term as used herein includes all integrin binding components described herein, including those with spacer elements GG and GA and those with preferred spacer elements.
  • the polycationic nucleic acid-binding component is any polycation that is capable of binding to DNA or RNA is retained.
  • from 4 to 100 cationic monomers may be present, for example, from 10 to 20, especially about 16.
  • An oligolysine is particularly preferred, for example, having from 3 to 100 lysine residues, for example, from 10 to 20, for example, from 13 to 19, for example, from 14 to 18, for example, from 15 to 17 residues, especially 16, 17 or 18 residues especially [K] ⁇ 6 , "K" denoting lysine.
  • the polycationic DNA or RNA-binding component may advantageously be linked or otherwise attached to the integrin-binding component .
  • a combined integrin-binding component/polycationic DNA or RNA-binding component may be referred to below as component "I".
  • a polycationic DNA or RNA-binding component may be chemically bonded to an integrin-binding component, for example, by a peptide bond in the case of an oligolysine.
  • the polycationic component may be linked at any position of the integrin- binding component.
  • Preferred combinations of integrin-binding component and polycationic DNA or RNA-binding component are an oligolysine, especially [K]i6, linked via a peptide bond to a peptide, for example, a peptide as described above.
  • a combined integrin-binding component/polycationic DNA or RNA-binding component is a component having an integrin- binding moiety that is the integrin-binding component as defined and described herein linked or otherwise attached to a polycationic DNA or RNA binding moiety that is a polycationic DNA or RNA component as defined and described herein.
  • the lipid component may be or may form a cationic liposome.
  • the lipid component may be or may comprise one or more lipids selected from cationic lipids and lipids having membrane destabilising or fusogenic properties, especially a combination of a cationic lipid and a lipid that has membrane destabilising properties.
  • a preferred lipid component (“L”) is or comprises the neutral lipid dioleyl phosphatidylethanolamine, referred to herein as "DOPE” .
  • DOPE has membrane destabilising properties sometimes referred to as “fusogenic” properties (Farhood et al . 1995).
  • Other lipids, for example, neutral lipids, having membrane destabilising properties, especially membrane destabilising properties like those of DOPE may be used instead of or as well as DOPE.
  • phospholipids having at least one long chain alkyl group for example, di(long alkyl chain) phospholipids may be used.
  • the phospholipid may comprise a phosphatidyl group, for example, a phosphatidylalkanolamme group, for example, a phosphatidyl-ethanolamine group.
  • a further preferred lipid component is or comprises the cationic lipid N- [1- (2 , 3 -dioleyloxy) propyl] -N, ,N- rimethyl- ammonium chloride, referred to herein as "DOTMA" .
  • DOTMA has cationic properties.
  • Other cationic lipids may be used in addition to or as an alternative to DOTMA, in particular cationic lipids having similar properties to those of DOTMA.
  • Such lipids are, for example, quaternary ammonium salts substituted by three short chain alkyl groups, and one long chain alkyl group.
  • the short chain alkyl groups may be the same or different, and may be selected from methyl and ethyl groups . At least one and up to three of the short chain alkyl group may be a methyl group.
  • the long alkyl chain group may have a straight or branched chain, for example, a di (long chain alkyl) alkyl group.
  • Another preferred lipid component is or comprises the lipid 2 , 3-dioleyloxy-N- [2- (spermidinecarboxamido) ethyl] -N,N- dimethyl-1-propanaminiumtrifluoridoacetate, referred to herein as "DOSPA".
  • Analogous lipids may be used in addition to or as an alternative to DOSPA, in particular lipids having similar properties to those of DOSPA. Such lipids have, for example, different short chain alkyl groups from those in DOSPA.
  • a preferred lipid component comprises DOPE and one or more other lipid components, for example, as described above.
  • Such mixtures form cationic liposomes.
  • An equimolar mixture of DOPE and DOTMA is found to be particularly effective.
  • Such a mixture is known generically as “lipofectin” and is available commercially under the name “Lipofectin”.
  • lipofectin is used herein generically to denote an equimolar mixture of DOPE and DOTMA.
  • Other mixtures of lipids that are cationic liposomes having similar properties to lipofectin may be used.
  • Lipofectin is particularly useful as it is effective in all cell types tested.
  • a further preferred lipid component comprises a mixture of DOPE and DOSPA. Such mixtures also form cationic liposomes.
  • a mixture of DOPE and DOSPA in a ratio by weight 3:1 DOSPA:DOPE is particularly effective.
  • Such a mixture, in membrane filtered water, is available commercially under the name "Lipofectamine” .
  • Mixtures comprising DOPE, DOTMA and DOSPA may be used, for example, mixtures of lipofectin and lipofectamine .
  • cationic lipids are available commercially, for example, DOTAP (Boehringer-Mannheim) and lipids in the Tfx range (Promega).
  • DOTAP is N- [1- (2 , 3-diolyloxy) propyl] -N,N,N- trimethylammonium methylsulphate.
  • the Tfx reagents are mixtures of a synthetic cationic lipid [N,N,N',N'- tetramethyl-N,N' -bis (2-hydroxyethyl) -2,3-di (oleoyloxy) -1,4- butanediammonium iodide and DOPE. All the reagents contain the same amount of the cationic lipid component but contain different molar amounts of the fusogneic lipid, DOPE.
  • lipofectin and lipofectamine appear to be markedly more effective as the lipid component in LID complexes of the present invention than are DOTPA and Tfx agents.
  • the effectiveness of a putative integrin-binding component, polycationic DNA or RNA-binding component, or of lipid component may be determined readily using the methods described herein.
  • the efficiency of transfection using an integrin-targeted transfection complex as described above as transfection vecotr is influenced by the ratio lipid component : integrin- binding component :DNA or RNA.
  • the optimal ratios can be determined simply by admixing the components in different ratios and measuring the transfection rate for that cell type, for example, as described herein.
  • a combination consisting of a pGL2 plasmid, which is a plasmid encoding luciferase (a reporter gene) under an SV40 promoter as DNA component (D) , [K] isGACRGDMFGCA [SEQ. ID.NO. :20] ( [K] ⁇ 6 -peptide 1) as a combined integrin- binding component/polycationic DNA binding component (I) , and lipofectin (DOPE:DOTMA 1:1 molar ratio) as the lipid component (L) was tested to find the optimal ratio of components.
  • a ratio L:I:D of 0.75:4:1 by weight corresponds to a molar ratio of 0.5 nmol lipofectin: 1.25 nmol [K] 16 -peptide 6: 0.25 pmol plasmid pGL2-control .
  • a ratio L:I:D of 0.75:4:1 by weight, or the corresponding molar ratio are preferred when lipofectin is used as the lipid component .
  • the optimal ratio was found to be 12 ⁇ g lipofectamine: 4 ⁇ g [K] ⁇ 6 -peptide 6 : 1 ⁇ g plasmid DNA or RNA.
  • a ratio of L:I:D of 12:4:1 by weight, or the corresponding molar ratio, is appropriate for lipofectamine-containing complexes.
  • Optimal ratios for other systems may be determined analogously.
  • Lipofectin and lipofectamine appear to be particularly effec- tive in enhancing transfection in the system described above. Lipofectin has the advantage that only very small amounts are required. Any side effects that may occur are therefore minimised. As indicated above, the optimal weight ratio of components L:I:D when using lipofectamine is 12:4:1. With lipofectin the optimal ratio is only 0.75:4:1.
  • a transfection complex as described above may be produced by admixing components (i) , (ii) , (iii) and (iv) .
  • the components may be admixed in any order, it is generally preferable that the lipid component is not added last.
  • lipid component lipid component
  • combined integrin- binding/polycationic DNA or RNA-binding component DNA or RNA component, for example, in the order: lipofectin, oligolysine-peptide component, DNA or RNA component.
  • EGTA For use with EGTA in vivo, it is preferable to mix the components as follows: mix the lipid component with the integrin binding/polycationic DNA. or RNA binding component; s mix the DNA or RNA with the EGTA; mix the two mixtures.
  • a transfection mixture comprising an integrin-binding compo- nent, a polycationic nucleic acid-binding component, and a lipid component may be used to produce a nucleic acid-containing transfection complex as described above by the incorporation of a nucleic acid with the mixture, for example, by admixture.
  • the transfection mixture may be used for the production of a complex which comprises, instead of the nucleic acid component, any other component that is capable of binding to the polycationic nucleic-acid binding component, for example, a protein.
  • transfection complex The individual components of a transfection mixture of the invention are each as described above in relation to the transfection complex.
  • the preferred components, preferred combinations of components, preferred ratios of components and preferred order of mixing, both with regard to the mixture and to the production of a complex, are as described above in relation to the transfection complex.
  • a transfection mixture preferably comprises an equimolar mixture of DOPE and DOTMA (lipofectin) as the lipid component and an oligolysine-peptide especially a [K] ⁇ 6 -peptide as a combined integrin-binding/nucleic acid-binding component .
  • the preferred molar ratio lipofectine:oligolysine-peptide is 0.75:4.
  • the present invention provides a method of transfecting cells, especially confluent cells or other slowly dividing or non-dividing cells that are in contact with each other, with a nucleic acid, which comprises contacting the cells in vi tro or in vivo with a receptor- targeted vector comprising a nucleic acid and with an agent that disrupts cell-cell junctions under conditions suitable for effecting transfection.
  • the present invention also provides a process for expressing a nucleic acid in host cells, especially confluent cells or other slowly dividing or non-dividing cells that are in contact with each other, which comprises contacting the host cells in vi tro or in vivo with a receptor-targeted vector comprising the nucleic acid and with an agent that disrupts cell-cell junctions under conditions suitable for effecting transfection and then culturing the host cells under conditions that enable the cells to express the nucleic acid.
  • the present invention further provides a process for the production of a protein in host cells, especially confluent cells or other slowly dividing or non-dividing cells that are in contact with each other, which comprises contacting the host cells in vi tro or in vivo with a receptor-targeted vector that comprises a nucleic acid that encodes the protein and with an agent that disrupts cell-cell junctions, under conditions suitable for effecting transfection, allowing culturing the host cells under conditions suitable for protein production, the cells to express the protein, and obtaining the protein.
  • the protein may be obtained either from the host cell or from the culture medium.
  • the present invention further provides confluent cells or other slowly dividing or non-dividing cells that are in contact with each other, transfected with a nucleic acid, and also the progeny of such cells .
  • the present invention further provides a disease model for use in testing candidate pharmaceutical agent, which comprises confluent cells or other slowly dividing or non- dividing cells that are in contact with each other, transfected with a nucleic acid suitable for creating the disease model.
  • candidate pharmaceutical agent which comprises confluent cells or other slowly dividing or non- dividing cells that are in contact with each other, transfected with a nucleic acid suitable for creating the disease model.
  • the present invention also provides a pharmaceutical composition which comprises (i) a receptor-targeted vector comprising a nucleic acid and (ii) an agent that disrupts cell-cell junctions, in admixture or conjunction with a pharmaceutically suitable carrier.
  • the composition may be a vaccine.
  • the present invention also provides a method for the treatment or prophylaxis of a condition caused in a human or in a non-human animal by a defect and/or a deficiency in a gene, which comprises administering to the human or to the non-human animal (i) a receptor-targeted vector comprising a nucleic acid suitable for correcting the defect or deficiency and (ii) an agent that disrupts cell-cell junctions in an amount effective for said treatment or prophylaxis.
  • the present invention also provides a method for therapeutic or prophylactic immunisation of a human or of a non-human animal, which comprises administering to the human or to the non-human animal (i) a receptor-targeted vector comprising an appropriate nucleic acid and (ii) an agent that disrupts cell-cell junctions in an amount effective for said therapeutic or prophylactic immunisation.
  • the present invention also provides a method of anti-sense therapy of a human or of a non-human animal, comprising anti- sense DNA administering to the human or to the non-human animal (i) a receptor-targeted vector comprising the anti- sense nucleic acid and (ii) an agent that disrupts cell-cell junctions in an amount effective for said anti-sense therapy.
  • the present invention also provides the use of (i) a receptor-targeted vector comprising a nucleic acid and (ii) an agent that disrupts cell-cell junctions for the manufacture of a medicament for the prophylaxis of a condition caused in a human or in a non-human animal by a defect and/or a deficiency in a gene, for therapeutic or prophylactic immunisation of a human or of a non-human animal, or for anti-sense therapy of a human or of a non- human animal .
  • a non-human animal is, for example, a mammal, bird or fish, and is particularly a commercially reared animal .
  • the nucleic acid, either DNA or RNA, in the vector is appropriate for the intended use, for example, for gene therapy, gene vaccination, or anti-sense therapy.
  • the DNA or RNA and hence the vector is administered in an amount effective for the intended purpose.
  • Conditions suitable for effecting transfection and for allowing cells to express protein are well known. Suitable conditions are described, for example, in the following Examples.
  • treatment and uses described above may be carried out by administering the respective vector, agent or medicament in an appropriate manner, for example, administration may be topical, for example, in the case of airway epithelia.
  • the present invention provides a kit comprising (i) a receptor-targeted vector comprising a nucleic acid and (ii) an agent that disrupts cell-cell junctions .
  • the present invention also provides a kit that comprises an agent that disrupts cell-cell junctions and the following items: (a) an integrin-binding component; (b) a polycationic nucleic acid-binding component, and (c) a lipid component.
  • a kit may further comprise (d) either a nucleic acid or a plasmid or vector suitable for the expression of a nucleic acid, the plasmid or vector being either empty or comprising the nucleic acid.
  • the components (a) to (d) kit are, for example, as described above in relation to a integrin-targeted transfection complex or a mixture as described above .
  • a kit generally comprises instructions, which preferably indicate the preferred ratios of the components and the preferred order of use or admixing of the components, for example, as described above.
  • a kit may be used for gene therapy, gene vaccination or anti-sense therapy.
  • it may be used for transfecting a host cell with a nucleic acid encoding a commercially useful protein i.e. to produce a so-called "cell factory”.
  • kits of the invention the components including the preferred components are, for example, as described above in relation to a complex of the present invention.
  • the present invention also provides a method for increasing the efficiency of transfecting confluent cells or other slowly dividing or non-dividing cells that are in contact with each other with a receptor targeted vector, which comprises treating the cells with an agent that disrupts cell-cell junctions.
  • the vector and the agent that disrupts cell- cell junctions are each as described above.
  • the vectors are non—viral vectors.
  • the confluent cells or other slowly dividing or non-dividing cells that are in contact with each other are cells that are substantially non-mitotic.
  • the vector is especially an integrin targeted transfection vector complex, as described above and in W098/53437, in particular one of the preferred complexes.
  • the integrin- binding component is preferably targeted at an integrin that is expressed abundantly in the tissue of interest.
  • ⁇ 5 ⁇ l integrins are expressed abundantly on bronchial epithelial cells, so an integrin-binding component directed at ⁇ 5 ⁇ l integrins is preferably used for transfection of such airway cells.
  • the polycationic nucleic acid binding component is preferably an oligolysine, as described above.
  • the lipid component is preferably capable of forming a cationic liposome, and preferably is or comprises DOPE and/or DOTMA, for example, an equimolar mixture thereof, or is or comprises DOSPA, for example, a mixture of DOPE and DOSPA, for example in the weight ratio DOPE:DOSPA of 1:3.
  • the rations between the components are preferably as described above, as is the order of mixing of the components.
  • the agent that disrupts cell-cell junctions is especially a calcium-binding agent, especially EGTA or maybe an antibody to a substance involved in cell-cell adhesion, for example, an anti-cadherin.
  • Targets for gene therapy are well known and include monogenic disorders, for example, cystic fibrosis, various cancers, and infections, for example, viral infections, for example, with HIV.
  • transfection with the p53 gene offers great potential for cancer treatment.
  • Targets for gene vaccination are also well known, and include vaccination against pathogens for which vaccines derived from natural sources are too dangerous for human use and recombinant vaccines are not always effective, for example, hepatitis B virus, HIV, HCV and herpes simplex virus.
  • Targets for anti- sense therapy are also known. Further targets for gene therapy and anti-sense therapy are being proposed as knowledge of the genetic basis of disease increases, as are further targets for gene vaccination.
  • tissue comprising confluent cells or other slowly dividing or non-dividing cells that are in contact with each other i.e. cells that are substantially non-mitotic.
  • the present invention enhances the transfection efficiency and hence the effectiveness of the treatment.
  • Transfection vector complexes described in W098/53437 and above have been demonstrated to transfect various different cell types, including endothelial and epithelial cells, and tumour cells. Transfection of all cell type's tested including cell types that are particularly resistant to transfection with most plasmid transfection vectors, for example, neuroblastoma cells, primary smooth muscle cells and cardiac myocytes, and haematopoieic cells has been achieved with high efficiency using transfection complexes of the present invention. This enables effective gene therapy, gene vaccination and anti-sense therapy without the previous restrictions as to cell type. For example, transfection with the p53 gene for cancer therapy has great potential but is currently limited by the range of cell types in which effective transfection can be achieved. The present invention enhances the transfection efficiency of such vectors when used to transfect confluent cells or other slowly dividing or non-dividing cells that are in contact with each other and hence the effectiveness of the treatment.
  • the effective transfection of primary smooth muscle cells and cardiac myocytes, which are particularly resistant to plasmid-mediated transfection, demonstrates that diseases and other pathological conditions affecting muscles and the cardiovascular system can now be treated by gene therapy.
  • One such condition is restenosis. After balloon angioplasty plaques reform in 30-50% of cases.
  • a gene that prevents proliferation of cells in blood vessel walls may be introduced using a complex of the present invention to reduce restenosis.
  • Haematopoietic cells are another cell type that is particularly resistant to plasmid-mediated transfection.
  • tranfection using a complex of the present invention which can exceed 60% , now enables gene therapy, gene vaccination and anti-sense therapy of diseases involving haematopoietic cells, including leukaemia and bone marrow stem cell disorders.
  • transfection of a cytokine gene may be used for adjuvant immunotherapy.
  • Complexes of the invention have been demonstrated to be effective vectors for intracellular transport and delivery of anti-sense oligonucleotides, which enables antiviral and cancer therapy.
  • complexes of the invention have been demonstrated to be effective for intracellular transport of very large DNA molecules, for example, DNA larger than 125kb, which is particularly difficult using conventional vectors. This enables the introduction of artificial chromosomes into cells.
  • the present invention enhances the transfection efficiency and hence the effectiveness of the treatment when confluent cells or other slowly dividing or non-dividing cells that are in contact with each other are treated.
  • Transfection of the airways for example, the bronchial epithelium demonstrates utility for gene therapy of, for example, cystic fibrosis and asthma.
  • Transfection of corneal endothelium demonstrates utility for treatment of eye disease affecting the cornea or corneal organ transplants, for example in glaucoma.
  • the present invention is particularly useful for enhancing the transfection efficiency and hence the effectiveness of the treatment of such epithelial and endothelial tissues.
  • the enhanced levels of transfection make the method of the invention particularly suitable for the production of host cells capable of producing a desired protein, so-called "cell factories".
  • cell factories For long-term production, it is desirable that the introduced nucleic acid is incorporated in the genome of the host cell, or otherwise stably maintained. That can be readily ascertained.
  • the range of proteins produced in this way is large, including enzymes for scientific and industrial use, proteins for use in therapy and prophylaxis, immunogens for use in vaccines and antigens for use in diagnosis.
  • Confluent cells may be used as tissue models, for example, of epithelial or endothelial tissue, for example, for use in testing of drugs.
  • tissue models for example, of epithelial or endothelial tissue, for example, for use in testing of drugs.
  • Such transfected cell cultures are part of the present invention, as are their uses in drug testing.
  • the present invention provides a method of testing drugs in a tissue model for a disease, wherein the tissue model comprises transgenic confluent cells obtained by transfecting confluent cells with a nucleic acid by contacting the cell with a receptor-targeted vector comprising a nucleic acid and with an agent that disrupts cell-cell junctions.
  • the present invention is especially useful with a receptor targeted vector that is capable of high efficiency targeted vector that is capable of high efficiency transfection.
  • the vector comprises four modular elements; an oligolysine, especially [K] ⁇ 6 , DNA or RNA-binding element; a high affinity integrin-binding peptide, for example, a peptide described herein; a DNA or RNA sequence, optionally in a plasmid, and optionally regulated by a viral promoter and an enhancing element; the cationic liposome DOTMA/DOPE (lipofectin) .
  • the combination of oligolysine-peptide/DNA or RNA complex with the cationic liposome formulation DOTMA/DOPE is a potent combination.
  • DOPE/DOSPA formulation may be used instead of or in addition to a DOTMA/DOPE formulation.
  • optimisation of variables associated with complex formation and the mode of transfection by LID complexes has been demonstrated.
  • analysis by atomic forces microscopy has been carried out to assess the structure of the complexes.
  • the level of transfection with LID (lipofectin/ [K] ⁇ e - peptide/plasmid) complexes is three to six fold higher than that with LKD (lipofectin/ [K] ⁇ 6 /plasmid) complexes prepared with the same charge ratios, or with LD (lipofectin/plasmid) complexes. This indicates that the integrin-targeting moiety, i.e. the peptide, is a significant factor in the transfection efficiency of those complexes.
  • Optimised LID transfection complexes contain only one seventh of the amount of lipofectin required for optimal transfection with LD complexes.
  • Transfections with low-ratio LD complexes that contain the same ratio of lipofectin to [K] i6-peptide/- plasmid as in optimal LID complexes but no [K] ⁇ 6 -peptide, did not transfect cells at all. This suggests that the role of lipofectin in LID complexes is to enhance transfection mediated by the integrin receptor-binding peptide.
  • LID and ID complexes both form spherical particles of similar sizes.
  • Optimal LD complexes formed a tubular network with some tubule-associated particles, which suggests a different type of cellular interaction and transfection mechanism from LID and ID transfections .
  • transfection by LID complexes were prepared containing a range of proportions of [K] ⁇ 6 and [K] i ⁇ integrin targeting peptide 6, [K] ⁇ 6 GACRRETAWACG [SEQ. ID.NO. :21] .
  • Transfection expression data indicate higher efficiencies with complexes in which increasing amounts of [K] lepeptide 6 replace [K] is and a dose-dependency on the amount of integrin-targeting (ligandbinding) domain i.e. peptide 6.
  • the ratio of components mixed together to form the optimal transfection complex is also informative as to the possible mechanism of lipofectin mediated enhancement.
  • the DOTMA element of lipofectin is cationic, which may enhance the activity of the complex, while DOPE may have the ability to destabilise the endosomal membrane (Farhood et al . , 1995) enhancing endosomal release of plasmid DNA or RNA.
  • the components of the LID complexes are mixed together in constant optimal ratios. It is assumed that the particles formed also contain these elements in the same proportions. Therefore, 3 nmol negative charge from plasmid DNA or RNA are associated with approximately 21 nmol positive charge from the [K] ⁇ 6 peptide.
  • Lipofectin however, provides only a further 0.25 nmol of positive charge. This suggests that, contrary to expectations, the enhancing effect of lipofectin in LID complexes is not charge related but may relate to the membrane destabilising effect of the DOPE component.
  • the complexes are formed electrostatically by random association of lipofectin, oligolysine-peptide and plasmid DNA or RNA.
  • the relative high proportion of oligolysine- peptide ensures a high proportion of integrin-targeting ligands per plasmid molecule.
  • Particles are formed that contain one or more plasmids, associated with thousands of oligolysine-peptides and, therefore, a very high concentration of integrintargeting ligands.
  • the particles due to the high density of ligands, have a" high avidity for integrins on cell surfaces, bind , and are internalised by a phagocytic process (Hart et al . , 1994) .
  • the vesicles fuse to form endosomes where, under acid conditions, the DOPE element contained within the particles mediates destabilisation of the endosomal membrane and subsequent plasmid release into the cytoplasm. Phagocytosed particles lacking lipofectin are degraded in the endosomes. Particles lacking the integrin-targeting moiety are less efficient at cell binding and internalisation.
  • Cystic fibrosis is the most common monogenic disorder in the Caucasian population. Morbidity is mainly associated with lung disease. CF is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) , a cell membrane channel that mediates secretion of chloride ions. Correction of this defect in the bronchial cells by CFTR gene transfer will correct the biochemical transport defect and, hence, the lung disease. Clinical trials so far have generated encouraging data but highlighted the need for more efficient, non-toxic vectors.
  • CFTR cystic fibrosis transmembrane conductance regulator protein
  • integrin-targeted transfection vector complexes comprising peptide 6 above, which is an ⁇ 5 ⁇ l-targeted peptide, linked to oligolysine comprising 16 lysines i.e. Ki6, (component I); lipofectin (component L) ; and a reporter gene (component D) were used with EGTA for transfection.
  • peptide 6 above which is an ⁇ 5 ⁇ l-targeted peptide, linked to oligolysine comprising 16 lysines i.e. Ki6, (component I); lipofectin (component L) ; and a reporter gene (component D)
  • Ki6 lysines
  • lipofectin component L
  • a reporter gene component D
  • Examples 1 to 14 relate to integrin-binding transfection vector complexes and their uses.
  • Examples 15 and 16 illustrate the transfection of confluent cells in vi tro and in vivo .
  • the Examples refer to the accompanying drawings, in which:
  • Figure 1 shows the effect of different amounts of lipofectin (DOTMA:DOPE) on the enhancement of transfection of ECV304 cells using a complex consisting of lipofectin, oligolysine- peptide 1 ( [K] leGACRGDMFGCA [SEQ . ID .NO. : 22] ) and plasmid pGL2.
  • Figure 2 shows the effect of different amounts of lipofectin on the enhancement of transfection of A375M, COS-7 and ECV-40 cells using a complex consisting of lipofectin, oligolysine- peptide 1 ( [K] leGACRGDMFGCA) and plasmid pGL2.
  • Figure 3 shows the effect of the order of mixing the components of a complex consisting of lipofectin (L) , oligolysine-peptide 1 ( [K] ⁇ 6 GACRGDMFGCA) (I) and plasmid pGL2 (D) on the enhancement of transfection of ECV40 cells.
  • L lipofectin
  • I oligolysine-peptide 1
  • D plasmid pGL2
  • Figure 4 shows a comparison of enhancement of transfection by lipofectin of complexes containing plasmid pGL2 and oligolysine-peptide 1 ( [K]i 6 GACRGDMFGCA, pep 1), or oligolysine- peptide 5 ( [K] leGACDCRGDCFCA [SEQ . ID .NO . : 22] , pep 5), or oligolysine-peptide 6 ( [K] ⁇ 6 GACRRETAWACG [SEQ. ID.NO.
  • Figure 5 shows the dose-dependency of a complex containing lipofectin, oligolysine-peptide 6 ( [K] ⁇ 6 GACRRETAWACG) and plasmid pGL2 on the availability of integrin-binding ligands.
  • Figure 6 shows the structure of various complexes, as determined using atomic force microscopy, the complexes being formed with different combinations of plasmid DNA (plasmid pGL2) , oligolysine-peptide ( [K] i6-peptide 6) and lipofectin as follows: A: [K] ⁇ 6 -peptide 6 and plasmid pGL2 ; B: [K] i6 - peptide 6, lipofectin and plasmid pGL2 ; C: lipofectin and plasmid pGL2, optimal ratio; D: lipofectin and plasmid pGL2, suboptimal ratio .
  • Figure 7 shows levels of expression of IL-12 48 hours after transfection of COS-7 cells and neuroblastoma cells lines IMR-32, KELLY and SHSY-5Y with a complex containing lipofectin, oligolysine-peptide 6 ( [K] ⁇ 6 GACRRETAWACG) and either two retroviral plasmid constructs encoding the two domains of IL-12 (MFGS-IL12) or one plasmid containing a fusion gene, Flexi-12 under a CMV promoter.
  • Figure 8 shows the effect of transfection with anti-sense oligonucleotides (AS) to the thrombin receptor (PAR-1) on thrombin induced proliferation of human foetal lung fibroblasts (HFL-1 cells) .
  • AS anti-sense oligonucleotides
  • PAR-1 thrombin receptor
  • Figure 9 shows the effect of transfection of haematopoietic cell lines HL60, PLB985, TF1 and U937 with LID complexes containing lipofectin, the reporter gene pEGFP-Nl and either [K] ⁇ e-peptide 6 (pep 6) or [K] i6-peptide 8 (GGCRGDMFGCA [SEQ. ID.NO. :8] pep 8) compared with untreated cells.
  • the percentage of GFP positive cells is determined using a fluorescence activated cell sorter.
  • Figure 10 shows GFP (green fluorescent protein) transfected confluent human airway epithelium cells.
  • Figure 10A shows the cells transfected by a transfection vector complex in the absence of EGTA
  • Figure 10B shows transfection in the presence of EGTA.
  • Figure 11 shows the effect of EGTA on transfection of mouse lung in vivo using luciferase as the reporter gene. Results are given in RLU per mg lung protein for transfection with no EGTA, with 100 mM EGTA and with 400 mM EGTA.
  • Figure 12 shows flow cytometry analysis on the transfection efficiency with pEGFP in subconfluent (A, B) and confluent (C, D) lHAEo- cells (A, C) and 2CFSMEO- cells (B, D) . High efficiency was observed in subconfluent cells but it dropped dramatically when the cells became confluent.
  • Figure 13 shows luminometric assay of the effects of EGTA (A, B) and anti-E-cadherin (C, D) on transfection.
  • the confluent (A) and subconfluent (B) lHAEo- cells were transfected with LID complexes after pre-treatment with EGTA (EGTA) or in the presence of EGTA (LID+EGTA) .
  • Significant enhancement effects were observed in confluent but not subconfluent cells with LID vector incorporated with EGTA (LID+EGTA) compared with either the EGTA pretreatment (EGTA) or the control transfection without EGTA (OptiMEM) .
  • Figure 14 shows flow cytometry analysis of EGTA effects on transfection efficiency (A-C) and the rate of BrdU incorporation (D-F) in confluent lHAEo- cells.
  • Cells were, transfected with pEGFP (A) or pEGFP plus EGTA (B) , or pEGFP plus EGTA and aphidicolin (C) .
  • cells were labelled with BrdU (D) or BrdU plus EGTA (E) , or BrdU plus EGTA and aphidicolin (F) .
  • D BrdU
  • E BrdU plus EGTA
  • F BrdU plus EGTA and aphidicolin
  • EGTA increased the efficiency to 31.7% (B) and this enhancement effect was still observed in the presence aphidicolin where the EGFP-positive cells accounted for 23.6% (C) . Meanwhile, EGTA showed no effects on cell proliferation with the rate of BrdU incorporation being 7.1% in confluent cells (D) , 6.5% in the presence of EGTA (E) , and 5.3% in the presence of EGTA and aphidicolin (F) .
  • Figure 15 shows sub-confluent lHAEo- cells: flow cytometry analysis of EGTA effects on transfection efficiency (A-C) and the rate of BrdU incorporation (D-F) .
  • Cells were treated in the same way as in the confluent cells shown in Figure 13.
  • Transfection efficiency was as high as 44.5% in sub-confluent cells (A) .
  • Similar efficiency of 45.6% was observed in the presence of EGTA (B) and there were still 24.8% cells being transfected in the presence of aphidicolin (C) .
  • EGTA decreased BrdU incorporation rate in sub-confluent cells as the proportion of BrdU- positive cells dropped from 43.0% (D) to about 23.9% after EGTA treatment (E) . 7.8% cells were labelled with BrdU in the presence of aphidicolin (F) .
  • Figure 16 shows double immunofluorescence for transgene ⁇ - Galactosidase (transgene expression shown by green cytoplasmic staining, irregular shapes) and BrdU labelling (shown by red nuclear staining, smaller rounder shapes) in subconfluent (A) and confluent (B, C) lHAEo- cells. Most of the subconfluent cells were labelled with BrdU and many of them expressed transgene simultaneously (irregular shape containing rounded shape) , shown in Figure 15A. Only few confluent cells were positive for ⁇ - Galactosidase while few were labelled with BrdU (B) . After
  • Figure 17 shows photomicrographs of confluent lHAEo- cells showing the effects of EGTA treatment.
  • the images in left- hand column (A, C, E, G) shows the cells before EGTA treatment and the right-hand column (B, D, F, H) are the cells after EGTA treatment.
  • Examples 1 to 12 illustrate the production of transfection complexes and, as comparative examples, illustrate the transfection of cells that are dividing.
  • Examples 13 to 15 illustrate the transfection of non-dividing and/or confluent cells.
  • the cell line COS-7 (monkey kidney epithelial cells) were maintained in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, Paisley, U.K.) supplemented with 10 % foetal calf serum (FCS) , L-glutamine, penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS foetal calf serum
  • ECV304 spontaneous transformed human umbilical vein endothelial cells
  • HT1080 fibrosarcoma cells and A375M melanoma cells were maintained in DMEM and 10% FCS.
  • IMR2 neuroblastoma cells were grown in DMEM F12 Nutrient Mix
  • the sequence of peptide 6, GACRRETAWACG was based on an ⁇ 5 ⁇ l-specific peptide from a phage display library (Koivunen et al., 1995).
  • the oligolysine-peptide [K] ⁇ 6 GACRRETAWACG was synthesised as follows:
  • Cleavage from the resin and deprotection of the peptide was achieved by treating the peptidyl-resin with 10 ml of a mixture containing 10 ml trifluoroacetic acid, 0.25 ml ethanedithiol, 0.25 ml triisopropylsilane at 20°C for two hours.
  • the peptide was precipitated using ice-cold diethyl- ether and then filtered through a fine sintered glass filter funnel under light vacuum.
  • the peptide precipitate was dissolved in 10% acetic acid/water solution and freeze dried.
  • the crude peptide was analysed by reverse phase HPLC and matrix assisted laser desorption ionisation time of flight mass spectroscopy.
  • Oligolysine-peptide 1 [K] ⁇ 6 GACRGDMFGCA and oligolysine- peptide 5 : [K] leGACDCRGDCFCA were obtained from Zinsser Analytic (Maidenhead, U.K.).
  • the plasmids pGL2, which contains a luciferase reporter gene (Promega, Madison, WI, U.S.A.) and pCMV ⁇ , which contains a ⁇ - galactosidase reporter gene (Clontech, Palo Alto, California, U.S.A.) were grown in Escherichia coli DH5a and purified, after bacterial alkaline lysis, on Qiagen resin columns
  • Plasmid solutions were adjusted to a concentration of 1 mg/ml and stored at 4°C.
  • transfection complexes Formation of transfection complexes Cells were seeded into 24-well plates at 5 x 10 4 cells per well then incubated overnight at 37°C in complete growth medium. The following day, transfection complexes were made from the following stock solutions, all prepared in OptiMEM (Life Technologies, Paisley, U. K.
  • lipofectin an equimolar mixture of the cationic lipid N- [1- (2, 3-dioleyloxy) propyl] -- N,N,N-trimethylammonium chloride (DOTMA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) , obtained as "Lipofectin” from Life Technologies, Paisley, U.K.) (1 mg /ml), pGL2-control (1 mg/lOO ml) and [K] ⁇ 6 /integrin-targeting peptide 1, 5 or 6 (0.1 mg/ml) .
  • DOTMA cationic lipid N- [1- (2, 3-dioleyloxy) propyl] -- N,N,N-trimethylammonium chloride
  • DOPE neutral lipid dioleoyl phosphatidylethanolamine
  • oligolysine-peptide (I) oligolysine-peptide
  • D plasmid DNA or RNA
  • L lipofectin
  • the protein concentration of each sample was then determined with Protein Assay Reagent (BioRad, Hercules, CA, U.S.A.) and luciferase enzyme activity expressed in terms of relative light units per milligram of protein (RLU/mg) .
  • LacZ assays ⁇ -galactosidase activity was detected by staining with X-gal. After washing with PBS cells were fixed to the plastic plates by addition of 0.5% glutaraldehyde in PBS for 20 minutes at
  • Atomic Forces Microscopy Atomic forces microscope analysis of transfection complexes was performed as described previously (Wolfert & Seymour, 1996) using an AFM-2, part of the NanoScope II (Digital Instruments, Santa, Barbara, U.S.A.). Transfection complexes of [K] i6-peptide 6/pGL2, with and without lipofectin, were prepared as described above except that water was used as the diluent for all components rather than OptiMEM.
  • Transfection complexes were prepared as described above in the Materials & Methods section. The complexes were made by mixing solutions of oligolysine-peptide 1 ( [K] l ⁇ GACRGDMFGCA) at 0.1 mg/ml in OptiMEM low serum tissue culture medium with a solution of lipofectin (DOTMA/DOPE cationic liposome as above) in a range of concentrations from 1 to 10 ⁇ g/100 ⁇ l in OptiMEM. Finally, the appropriate amount of pGL2-control plasmid DNA (0.1 mg/ml) was added and mixed by repeated pipetting.
  • the ratio of mixing of each component was a constant 4 ⁇ g of oligolysine-peptide per ⁇ g of DNA, while the proportion of lipofectin varied from 1 to 10 ⁇ g per ⁇ g of DNA.
  • ECV304 cells were transfected with the complexes as described above, incubated for 48 hours then assayed for luciferase expression as described above. The results are shown in Figure 1.
  • Example 2 Effect of dif erent amounts of lipofectin on transformation in three different cell lines Experiments were then performed to refine the optimal amount of lipofectin in LID transfection complexes using three different cell lines A375M (melanoma cells) , COS-7 (monkey kidney epithelial cells) and ECV304 (human umbilical cord endothelial cells) .
  • A375M melanoma cells
  • COS-7 monkey kidney epithelial cells
  • ECV304 human umbilical cord endothelial cells
  • Transfection complexes were made as described in Example 1 but using a narrower range of amounts of lipofectin.
  • Lipofectin/oligolysine-peptide/DNA complexes were prepared with constant amounts of [K] ⁇ 6 -peptide 1 ( [K] X6 GACRGDMFGCA) (4 ⁇ g) and pGL2 (1 ⁇ g) plasmid DNA and a range of lipofectin amounts (1 to 2.5 micrograms) .
  • Complexes were used to transfect A375M, COS-7 and ECV304 cells, which were then harvested two days later for luciferase expression analysis.
  • a mixing ratio L:I:D of 0.75:4:1 by weight corresponds to a molar ratio of 0.5 nmol lipofectin: 1.25 nmol oligolysine- peptide 1: 0.25 pmol pGL2-control .
  • the molar charge of each component is 0.5 moles positive charge per mole lipofectin, seventeen moles positive charge per mole [K] i ⁇ -peptide 1 and 12,000 moles negative charge per mole of pGL2 (6 kb) . Therefore, in the optimal transfection complex, 3 nmol of negative charge from the plasmid is mixed with 21 nmol of positive charge from oligolysine-peptide 1 and 0.25 nmol positive charge from lipofectin.
  • transfections were performed with complexes made by adding the components of the complexes in different orders. All combinations were prepared with the same amounts and concentrations of the components (1 ⁇ g pGL2 plasmid DNA, 0.75 ⁇ g of lipofectin and 4 ⁇ g of oligolysine- peptide 1 ( [K] isGACRGDMFGCA) . Transfections were performed in ECV304 cells and luciferase activity was assessed as described above.
  • Cells were transfected with optimised oligolysine- peptide/lipofectin/pCMV ⁇ complexes as described in Examples 1 and 2 prepared in the order of mixing LID but using pCMV ⁇ as the plasmid vector (component D) instead of pGL2.
  • the cells were stained for ⁇ -galactosidase activity with X-gal as described above.
  • a number of cell types, A375M, COS-7 and ECV304 displayed transfection efficiencies of 50 to 100% compared to 1 to 10% achieved with oligolysine-peptide/DNA complexes alone. This represents a very significant improvement in transfection efficiency.
  • Example 5 Comparison of enhancement with lipofectin and with different oligolysine-peptides
  • duplicate sets of complexes were formed with plasmid pGL2 and one of the following: oligolysine-peptide 1 ( [K] ⁇ 6 GACRGDMFGCA, pep 1), oligolysine-peptide 5 ( [K] ⁇ 6 GACDCRGDCFCA, pep 5), oligolysine-peptide 6 ( [K] ⁇ 6 GACRRETAWACG, pep 6), and [K] 16 .
  • One set of complexes also contained lipofectin (lip) , the other was without lipofectin.
  • a control complex containing plasmid pGL2 with lipofectin and [K] l ⁇ lysine-peptide 1 in a ratio by weight of 4:1 was prepared.
  • LID complexes were prepared with constant amounts of plasmid pGL2-control and lipofectin, and a range of combinations of [K] i ⁇ -peptide 6 and [K]i6- A total of 40 ⁇ g of [K] 16 -peptide was used, consist- ing of 1, 5, 10, 20, 35, 39 ⁇ g of [K] 16 -peptide 6 made up to 40 ⁇ g with [K] 16 .
  • Transfections were performed as described in Example 1 and luciferase assays performed after 48 hours. The results are shown in Figure 5. Transfection efficiency demonstrated an apparently exponential increase with increasing amounts of oligolysine-peptide 6, and, therefore, a dose-dependent response to the amount of available integrin-binding ligands.
  • Atomic force microscopy experiments were performed to determine and compare the structures formed by mixing 4 ⁇ g [K] ⁇ S peptide 6 and 1 ⁇ g pGL2-control plasmid DNA (ID complexes) .
  • LID complexes were formed from [K] i6-peptide 6 (4 ⁇ g) /lipofectin (0.75 ⁇ g) /DNA (1 ⁇ g) in the order LID which was shown to yield optimal transfection results.
  • Lipofectin/DNA complexes (LD) were formed at two different ratios; an optimal transfection ratio of 5 ⁇ g lipofectin per microgram of pGL2 and the same ratio as used in LID complexes, 0.75 ⁇ g lipofectin per microgram of plasmid.
  • ID complexes composed of oligolysine-peptide 6 and plasmid DNA, were examined initially by AFM within fifteen minutes of mixing the two components.
  • the complexes formed particles of low polydispersity which, on the mica coverslips, had a diameter of approximately 200 nm.
  • a computer-generated contour map revealed that the particles formed were of irregular conical shape.
  • LID complexes assessed by AFM formed particles of a similar size and shape to ID complexes. The additional lipofectin did not, apparently, disrupt the particles.
  • LD com- plexes however, formed at the 5:1 ratio appeared as a network of tubes with occasional particles associated with the tubes.
  • LD complexes formed at the lower ratio (0.75:1) appeared to be short tubular structures. LD complexes formed at this lower ratio were inactive in transfection experiments.
  • LID complexes formed as above were also analysed by AFM after standing overnight. Particles were now smaller in size with diameters of approximately 50-100 nm suggesting that the particles had compacted. Computer-generated computer maps represented these particles as regular conical structures. The cones were measured and their volumes were calculated. The spheres which the particles are predicted to form when free in solution were then calculated to be 20 to 60 nm in diameter. In transfection experiments with pGL2 the compact particles formed overnight in water yielded luciferase expression results approximately twice as high as the freshly made complexes.
  • the three human neuroblastoma cell lines and COS-7 cells were then transfected using the same LID complex with, instead of the reporter gene, one of two different IL-12 expressing vectors.
  • One vector expresses a fusion protein of the two chains of IL-12, p35 and p40, (Flexi-12; Anderson et al . 1997) This fusion is regulated by a CMV promoter.
  • the second IL-12 expression system consists of two retroviral constructs MFGS-p35 and MFGS-p40, which are retroviral plasmid constructs encoding the two separate chains of interleukin-12 (IL-12) . Both genes are regulated' by the retroviral long terminal repeats (LTRs) .
  • the vectors were obtained from Professor Mary Collins, UCL, London.
  • transfection system of the present invention is suitable for use in a vaccine for neuroblastoma, an important childhood malignancy, and also for vaccines against other cancers .
  • Tissue cultures of rat primary smooth muscle cells (aortic smooth muscle cells) and cardiac myocytes were prepared according to standard methods (Blank et al . 1988) .
  • An LID complex comprising lipofectin, [K] ⁇ 6 -peptide 6 and GFP as a reporter gene in the optimal LID ratio and mixing order was prepared as described in the Materials and Methods section and the Examples above.
  • the tissue cultures were transfected with the LID complex as described in the Material and Methods section above. Fluorescing imaging of GFP-expressing cells demonstrates transfection efficiency in excess of 50%.
  • transfection complex of the present invention achieved transfection efficiencies in excess of 50%, thus demonstrating the utility of the complexes for treatment of diseases affecting muscle, including smooth muscle and cardiac muscle .
  • Example 10 Transfections with high molecular weight constructs
  • transfection complex of the present invention Different size constructs can be delivered with the transfection complex of the present invention.
  • a fibroblast culture was transfected as described in the Materials and Methods section with an LID complex comprising [K] ⁇ 6 -peptide 6, lipofectin and a 130 kB DNA construct.
  • the complex comprising the LID components in the optimal ratio and mixing order, was prepared as described in the Methods and Materials section and Examples above. Transfection was achieved with 2- 3% efficiency.
  • Cellular process associated with the enhanced integrin- mediated internalisation of DNA using a complex of the present invention are more closely related to phagocytosis than endocytosis and are thus particularly suited to the delivery of complexes containing very large DNA molecules.
  • Example 11 Transfection with anti-sense DNA Thrombin stimulates proliferation of human lung fibroblasts.
  • Thrombin-treated human lung fibroblasts HFL-1 cells proliferated 53% in response to thrombin.
  • HFL-1 cells 24 hours before treatment with thrombin, HFL-1 cells were treated with an LID complex comprising [K] ⁇ 6 -peptide 6, lipofectin and a 20-mer antisense oligonucleotide directed against the thrombin receptor PAR-1 in the optimal ratio and mixing order prepared as described in the Materials and Methods section and the Examples above.
  • the antisense oligonucleotide-containing complex was in contact with the cells for 4 hours. 24 hours after the start of the treatment with the complex, treatment with thrombin was carried out .
  • Haematopoietic cells are particularly resistant to transfection with most plasmid-mediated vectors.
  • LID complexes were prepared as described in the Material and Methods section and Examples above using lipofectin and [K] i 6 -peptide 6, which targets ⁇ 5 ⁇ l integrins, and pEGFP-Nl (Promega) as reporter gene. Complexes were prepared analo- gously substituting [K] ⁇ 6 -peptide 8 ( [K] i 6 GACQIDSPCA
  • Example 13 Transfection of confluent human airway epithelial cell in vi tro in the presence of EGTA
  • HAE Human airway epithelial cells
  • type HEAO- were seeded at 80% into 24 well plates.
  • the cells were grown in Modified Eagle Medium (MEM) obtained from Life Technologies (Paisley, Scotland) supplemented with 10% foetal calf serum and 2 mM L- glutamine to form cultures of confluent cells. Subconfluent cultures of the same cells were also prepared.
  • MEM Modified Eagle Medium
  • reporter genes were a green fluorescent protein
  • GFP GFP reporter gene
  • pEGFP-Nl plasmid pEGFP-Nl
  • the luciferase reporter gene was used in the form of construct pCILuc consisting of the firefly luciferase gene (Luc) inserted into the expresssion vector pCI (Promega, Southampton, England)
  • the oligolysine-peptide component I comprised the integrin-targeted peptide CRRETAWACG, which targets the integrin ⁇ 5 receptor, with a spacer XSXGA (X: ⁇ amino hexanoic acid), and the oligolysine [K]i6, giving the sequence [K] ⁇ 6 XSXGACRRETAWACG (Zinsser Analytic, Maidenhead, UK) .
  • Transfection vector complexes were prepared as described in the Material and Methods section, see also Example 15. The complexes were prepared using 4 ⁇ g of the peptide, 0.75 ⁇ g of lipofectin and 1 ⁇ g of the reporter gene. All components and the vector complex were made up in OptiMEM (Life Technologies, Inc.). EGTA (Sigma) at a concentration of 100 mM in water was added to the transfection vector complex preparation at one hundredth of the volume of the vector complex, giving a final EGTA concentration of 1 mM.
  • the EGTA/vector complex mixture was added to the cultures, which were then harvested two days later. As controls, cultures were treated with OptiMEM medium alone and with the transfection vector complex without EGTA.
  • transfection was carried out in the presence of aphidicolin, which inhibits DNA polymerase- ⁇ , which prevents transition of the cells from GI to S phase.
  • aphidicolin which inhibits DNA polymerase- ⁇ , which prevents transition of the cells from GI to S phase.
  • Aphidicolin Sigma, Poole, Dorset, England
  • Figure 10 shows clearly the influence of EGTA in the increase in the number of fluorescent cells transfected with the vector in the presence of EGTA (Figure 10B) compared with the vector alone, as shown in ( Figure 10A) .
  • Bromodeoxyuridine (BrdU) labelling enables assessment of cell proliferation.
  • BrdU kits are available from Zymed Laboratories, Inc., South San Francisco, California, U.S.A.. BrdU was added to the cells at the same time as the transfection reagent and was maintained throughout the rest of the experiment until cell harvesting.
  • the results of BrdU labelling of sub-confluent and confluent HAE cells is given in Table 3 below. TABLE 3
  • Example 14 Transfection of mouse lungs in vivo in the presence of EGTA
  • the protocol is different from that for the in vi tro experiments described in Example 13, because of the importance of minimising the final volume of the vector to be administered.
  • Lipid (lipofectin) (1 mg/ml) and peptide 6 (GACRRETAWACG) (1 mg/ml) were mixed in one tube and pCILuc Plasmid DNA (1 mg/ml) and EGTA in phosphate buffer saline (PBS) were mixed together in another tube then the contents of the two tubes were mixed so that the final ratios were 0.75 ⁇ g lipid to 1 ⁇ g DNA and a final concentration of 100 mM EGTA (or no EGTA for the control) .
  • the protocol was repeated to give vector complexes having a final EGTA concentration of 400 mM.
  • Each mouse received 8 ⁇ g of plasmid DNA in a volume of 50 ⁇ l .
  • the vector/EGTA mixture was administered to the mouse lung as described in Example 9 for rats, by intra-tracheal instillation.
  • mice were killed by cervical dislocation and the lungs perfused via the inferior vena cava with heparinised PBS until free of blood.
  • Cell lysis buffer Promega, Southampton, England
  • the tissue was then homogenised two times, on ice, for 30 seconds each time (Polytron PT10-35, Philip Harris, Nottingham, England) .
  • Lung homogenates were centrifuged at 10,000 x g at 4 C for 10 minutes, then 20 ⁇ l of the supernatant were added to 100 ⁇ l of luciferase assay buffer (Promega) and luminescence measured in a luminometer (TD-20/20; Steptech Instruments, Stevenage, England). All results were repeated in triplicate and the mean calculated.
  • 2CFS Meo- cells are immortalised, SV-40 T antigen transformed cells obtained from Dr D. Gruenert, UCSF.
  • the lHAEo- cells retain the morphologic and functional characteristics of epithelial cells and have been used in many different studies, see (Boussat et al (2000) , Cozens et al (1992a) .
  • the 2CFSMEo- cells are ⁇ F508 heterozygous submucosal epithelial cells, see (Canonico et al (1996) , Cozens et al (1996) .
  • Both cell lines were maintained in a humidified atmosphere of 95% air and 5% C0 2 at 37°C in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 2 mM .L- glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (Sigma, Poole, UK) in 75cm 2 culture flasks.
  • MEM Eagle's minimum essential medium
  • Cells were seeded on transwell inserts of 0.4 ⁇ m pore size and 12 mm diameter (Costar, Bucks, UK) at a density of 10 5 cells/0.5ml/insert and were left to grow until fully confluent in about 3 days.
  • apical medium was then removed and growth medium was added basolateraly to leave the cells growing at an air- liquid interface.
  • Confluent cells were grown for further 10- 14 days and were monitored with an ohmmeter (EVOM; World Precision Instruments, Stevenage, UK) until the transepithelial resistance was greater than 300 ⁇ -cm before transfection.
  • EVOM World Precision Instruments, Stevenage, UK
  • the cells were seeded on coverslips in 24-well plates at a density of 10 5 cells/ml/well and were left to grow for about 10-14 days until fully confluent .
  • the state of confluence and development of tight junctions were also determined by the immunofluorescent detection of occludin, the structural and functional component of tight junctions (detailed below) .
  • Sub-confluent, proliferating cells were prepared by growing the cells for less then 3 days in growth medium prior to transfection.
  • Plasmid DNA Three different plasmid DNA controls were used in this study. Plasmid pCI-Lux was prepared by subcloning a luciferase gene from pGL3 control (Life Technologies, Paisley, UK) into the eukaryotic expression vector pCI (Promega, Southampton, UK) .
  • Plasmid pCIK-LacZ containing cytoplasm-located bacterial ⁇ - Galactosidase gene, was obtained from Dr. Steve Hyde (Oxford) .
  • the plasmid pEGFP was commercially available (CLONTECH Laboratories UK Ltd., Hampshire, UK). All of these genes were driven by CMV promoter.
  • the plasmids were amplified in Escherichia coli DH5 , lysed with alkaline and prepared using an endotoxin-free kit (Qiagen, Crawley, UK) .
  • the DNA was washed with 70% ethanol and dissolved in water.
  • the concentration of DNA was spectrophotometrically determined by its absorbance at A 2 6o and the purity was assessed with the ratio of A 26 o/A 2 8o.
  • LID complexes and "LID vectors” hereafter, were prepared in a procedure similar to that described in Example 1. Briefly, stock solutions of the three components of the LID vector complex were prepared first. Plasmid DNA (component D) was diluted in cell culture medium OptiMEM (Life Technologies, Paisley, UK) at a concentration of 0.01 ⁇ g/ ⁇ l .
  • the oligolysine-peptide component I comprised the integrin-targeted peptide CRRETAWACG, which targets the integrin ⁇ 5 receptor, with a spacer XSXGA (X: ⁇ amino hexanoic acid) , and the oligolysine [K] ⁇ s, giving the sequence [K] ⁇ 6 XSXGACRRETAWACG (Zinsser Analytic, Maidenhead, UK) .
  • the peptide component I was dissolved in Opti-MEM at 0.1 mg/ml.
  • DOTMA cationic lipid N- [1- (2, 3-dioleyloxy) propyl] -n,n,n- trimethy1ammonium chloride
  • DOPE neutral lipid dioleoyl phosphotidy
  • the complexes were then left 30 minutes at room temperature before being diluted to a final concentration of l ⁇ g of DNA in 0.3 ml of Opti-MEM in transwells, or in 0.5 ml for 24-well plate.
  • the cells were incubated with the complexes for 4 hours at 37°C followed further culture in growth medium for 24 to 48 hours before being harvested for the assay of transgene expression.
  • EGTA-formulation protocol LID+EGTA
  • EGTA was added to the OptiMEM containing LID complexes, at a final concentration of 2 mM.
  • OptiMEM contains 0.9 mM CaCl 2 which is of half the level of conventional mediums.
  • EGTA was first dissolved in PBS (pH7.3) to make a stock solution of 200 mM. After preparing the LID complexes, EGTA stock solution was added to the complexes to achieve a final concentration of 2 mM. The transfection incubation was performed in the presence of EGTA but was replaced with complete growth medium after 4 hours.
  • Reporter Lysis Buffer Promega, Southampton, UK
  • Cell-free lysates were prepared by centrifugation at 2000 rpm for 5 minutes at 4°C.
  • the activity of luciferase was assayed with a luciferase assay kit (Promega, Southampton, UK) on a Lucy-1 plate-reading luminometer (Anthos, Salzburg, Austria) .
  • the protein concentration of each sample was determined with Protein Assay Reagent (BioRad Laboratories, Hertfordshire, UK) by measuring absorbance at 595 nm and the specific activity of luciferase was expressed as relative light unit per milligram protein (RLU/mg protein) .
  • Transfection efficiency by flow cytometry analysis of green fluorescent protein Cells transfected with pEGFP were washed twice with PBS, harvested by digesting with trypsin-EDTA and fixed in 4% paraformaldehyde. The cells were then analysed by fluorescence-activated cell sorting (FACS) with a FACS sorter EPICS XL (Beckman Coulter Inc, Buckinghamshire, UK) . Fluorescence for EGFP was measured at wavelength of 525+20 nm. Ten thousand cells were examined for the percentage of EGFP-positive cells by determining the percentage of highly fluorescent cells and subtracting the fluorescence of the untransfected control cells.
  • FACS fluorescence-activated cell sorting
  • Cell proliferation was assessed by incorporating 5-Bromo-2'- deoxy-uridine (BrdU) into cellular DNA detected by immunofluorescent staining using a monoclonal antibody.
  • a BrdU Labelling and Detection Kit (Roche, Basel, Switzerland) was used, which contains the BrdU labelling reagent and the monoclonal mouse anti-BrdU. While the confluent cells were transfected with EGTA-formulated LID containing pEGFP for analysis of transfection efficiency, a control group was transfected with the same protocol but with 10 ⁇ M BrdU in the transfection medium for analysis of cell proliferation.
  • aphidicolin a cell-proliferation inhibitor blocking the cell cycle in S-phase, was added to the cells to observe the effects on transfection efficiency.
  • Aphidicolin was added to the growth medium (10 ⁇ g/ml) for 24 hours before transfection and maintained throughout the 4- hour transfection and the following 24 h period of incubation until the cells were harvested.
  • FITC fluorescein
  • Peptide-12 was labelled with FITC from a labelling kit (Calbiochem, San Diego, USA) and purified by dialyzing in PBS overnight, according to the manufacturer's instruction.
  • the ability of FITC-conjugated peptide-12 to form complexes with plasmid DNA was determined by retardation test, in which the electrophoresis migration of DNA is retarded by forming complexes with the cationic peptide as described previously (Hart et al . , 1995).
  • the confluent cells in transwells were first incubated with 2 mM EGTA in Opti-MEM for 30 minutes at 37°C to disrupt tight junctions and then cooled for 20. minutes at 4°C.
  • the LID complexes prepared with FITC-labelled peptide-12 were also cooled to 4°C for binding assay.
  • the cells were then transfected with apically added LID-FITC for 60 minutes at 4°C, at which the complexes would only bind to cell surface and no internalisation would occur, see (Chu et al (1999), Cornelissen et al (1997) , Im et al (1986) .
  • the surface binding of the LID complexes was further differentiated from the internalised LID by incubating the cells with 1 ⁇ g/ml crystal violet (Sigma, Poole, UK) for 10 minutes at 4°C, to quench the fluorescence of surface-bound LID complexes, see (Ma et al (1997) , Van Amers & Van Strijp (1994) .
  • the mean numbers of fluorescent particles were then counted in 10 random microscope fields and comparison was made between the data obtained from EGTA- treated or untreated cells.
  • the ⁇ - galactosidase-transfected and BrdU-labelled (as detailed above in Assay of cell proliferation) cells were fixed with 70% ethanol in 50 mM glycine buffer, pH 2.0, for 30 minutes at -20°C.
  • the cells were then incubated with a mixture of two primary antibodies, the rabbit anti- ⁇ -galactosidase at 1:200 (Chemicon International Inc, Harrow, UK), and the mouse anti- BrdU at 1:10 (BrdU Labelling and Detection Kit; Roche, Basel, Switzerland) , followed by incubation with a mixture of FITC- conjugated goat anti-rabbit IgG and texas red labelled horse anti-mouse IgG (Vector Laboratories, Peterborough, UK) . Transwell inserts were then excised from the plastic holder and mounted on glass slides with aqueous mountant Vectashield (Vector Laboratories, Peterborough, UK) .
  • the EGTA-treated cells were immunostained with a rabbit polyclonal antibody against tight junction protein occludin (Zymed, San Francisco, USA) diluted 1:10 with PBS.
  • the antibody is specific to the C- terminal 150 amino acids of human occludin and has been used in many other studies, see (Jou et al (1998) , Martin-Padura et al (1998) .
  • the confluent cells on transwells were incubated with 2 mM EGTA in Opti-MEM for 30 minutes at 37°C and fixed in methanol for 20 minutes at 4°C.
  • the cells were immunostained with anti-occludin for 60 minutes, followed by incubation with a fluorescein labelled goat anti-rabbit IgG (Vector Laboratories, Peterborough, UK) for 30 minutes at 37°C.
  • the inserts were then excised and mounted on slides with Vectashield. Images were observed and captured with an inverted fluorescence microscope (Olympus 1X70, Olympus Optical Company, London, UK) for transwell and a confocal microscope (Leica TCS SP, Exton, PA, USA) for excised inserts.
  • the poor transfection efficiency of confluent cells could be due to 1) the lack of availability of receptors on basolateral surfaces, or 2) the low rate of cell proliferation and the inability of plasmid DNA to enter the nucleus across the nuclear envelop.
  • the first hypothesis was tested by treating the confluent epithelial cells with two agents known to disrupt cell junctions, EGTA and anti- cadherin monoclonal IgG, to augment gene transfer as reported previously for viral vectors, see Wang et al J Virol (1998) ; 72: 9818-9826 & Walters et al (1999) .
  • Cells were transfected with pEGFP (A) or pEGFP plus EGTA(B) , or pEGFP plus EGTA and aphidicolin (C) .
  • cells were labelled with BrdU (D) or BrdU plus ECTA (E) , or BrdU plus EGTA and aphidicolin (F) .
  • FIG. 16 shows photomicrographs of confluent lHAEo- cells showing the effects of EGTA treatment.
  • the images in left-hand column (A, C, E, G) shows the cells before EGTA treatment and the right-hand column (B, D, F, H) are the cells after EGTA treatment.
  • Confluent airway epithelial cells are almost entirely mitotically quiescent. As evident in this Example, the proliferating BrdU-labelled cells accounted for about 43% of subconfluent but only 7% in confluent cells. Reduced cell proliferation would result in a low efficiency of transfection as reported by Fasbender that cells in mitosis (BrdU positive) were much more likely to express transgene than BrdU-negative cells, see (Fasbender et al (1997) and, by Wilke that growth-arrested cells were less efficiently transfected, see (Wilke et al (1996) . The present study also showed the transfection was much more inefficient in confluent cells than in sub-confluent cells (8% vs. 44%). Increasing the rate of cell proliferation, therefore, would enhance gene transfer to airway epithelial cells as observed by transfecting the freshly seeded cells (Fasbender et al
  • EGTA was found to be highly effective for promoting access of the LID vector to basolateral receptors, enhancing transfection efficiency with low toxicity, see (Wang et al (1998) , Bals et al (1999) , Chu et al (1999) .
  • the results presented in this Example confirm that the efficient gene transfer to non-proliferating human airway epithelial cells using the synthetic nonviral vector LID, with adjuvant of a calcium chelator EGTA which transiently disrupted tight junctions to allow increased binding of LID complexes.
  • No induction of cells proliferation was observed by EGTA treatment and most of transgene-positive cells were non-proliferative .
  • Fernandez MA Muno-Fernandez MA, Fresno M. Involvement of ⁇ l integrins in the binding and entry of Trypanosoma cruzi into human macrophages . European J of Immunology 1993;23:552-557. 7. Wickham TJ, Filardo EJ, Cheresh DA, Nemerow GR.
  • Integrin av ⁇ 5 selectively promotes adenovirus mediated cell membrane permeabilization. J Cell Biology 1994;127 (1) :257-264.
  • Mitosis enhances transgene expression of plasmid delivered by cationic liposomes. Biochim Biophys Acta, 1445, 53-64.
  • Cadherins a molecular family important in selective cell-cell adhesion. Annu . Rev. Biochem . 59: 237-252.
  • junctional adhesion molecule a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J. Cell Biol . 142: 117-127.

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Abstract

L'invention concerne la transfection de cellules confluentes ou autres cellules à division lente ou sans division, en contact mutuel, par le biais d'un acide nucléique dépourvu de vecteur ciblé sur récepteur non viral, sachant que cette transfection peut être améliorée par l'utilisation concurrente d'un agent qui défait les liens entre cellules, en particulier l'EGTA. En particulier, le vecteur est un complexe à vecteur de transfection ciblant l'intégrine qui comprend (i) un acide nucléique, notamment un acide nucléique codant une séquence visée, (ii) un composant de liaison avec l'intégrine, notamment un peptide ciblant l'intégrine, (iii) un composant de liaison avec un acide nucléique polycationique, notamment une oligosine, et (iv) un composant lipide, notamment DOPE, DOTMA, DOSPA ou des combinaisons de ces éléments. L'invention concerne par ailleurs diverses applications du procédé de transfection amélioré qui est décrit.
EP01931977A 2000-05-30 2001-05-30 Procede ameliore de transfection Withdrawn EP1285081A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
GB0013090 2000-05-30
GB0013090A GB0013090D0 (en) 2000-05-30 2000-05-30 Intergrin-targetting vectors having enhanced transfection activity
GB0013089A GB0013089D0 (en) 2000-05-30 2000-05-30 Improved methods of transfection
GB0013089 2000-05-30
US28741001P 2001-05-01 2001-05-01
US287410P 2001-05-01
PCT/GB2001/002396 WO2001092543A2 (fr) 2000-05-30 2001-05-30 Procede ameliore de transfection

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FR2860236B1 (fr) * 2003-09-25 2006-01-06 Theraptosis Peptides possedant notamment une activite anti-angiogenique et leurs applications en therapeutique
CN102089320B (zh) 2008-01-24 2015-11-25 埃斯佩兰斯医药公司 溶解结构域融合构建体及其制备和使用方法
EP3468609A1 (fr) * 2016-06-09 2019-04-17 CureVac AG Supports cationiques destinés à l'administration d'acides nucléiques
EP3468613A1 (fr) 2016-06-09 2019-04-17 CureVac AG Supports hybrides pour cargo d'acides nucléiques

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CA2410419A1 (fr) 2001-12-06
WO2001092542A2 (fr) 2001-12-06
JP2003534804A (ja) 2003-11-25
AU2001260467A1 (en) 2001-12-11
WO2001092543A3 (fr) 2002-09-12
AU2001258659A1 (en) 2001-12-11
WO2001092542A3 (fr) 2003-05-30
WO2001092543A2 (fr) 2001-12-06
WO2001092542A8 (fr) 2003-12-24

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